U.S. patent application number 12/004502 was filed with the patent office on 2009-02-26 for moraxella catarrhalis protein, nucleic acid sequence and uses thereof.
This patent application is currently assigned to Emergent Product Development Gaithersburg, Inc.. Invention is credited to Ulrich F. Tillmann, Kenneth Tucker.
Application Number | 20090053292 12/004502 |
Document ID | / |
Family ID | 22595761 |
Filed Date | 2009-02-26 |
United States Patent
Application |
20090053292 |
Kind Code |
A1 |
Tucker; Kenneth ; et
al. |
February 26, 2009 |
Moraxella catarrhalis protein, nucleic acid sequence and uses
thereof
Abstract
The invention discloses the Moraxella catarrhalis outer membrane
protein polypeptide and polypeptides derived therefrom
(collectively "OMP21"), nucleotide sequences encoding said OMP21,
and antibodies that specifically bind OMP21. Also disclosed are
pharmaceutical compositions including prophylactic or therapeutic
compositions, which may be immunogenic compositions including
vaccines, comprising OMP21, antibodies thereto or nucleotides
encoding same. The invention additionally discloses methods of
inducing an immune response to M. catarrhalis and OMP21 in an
animal, preferably a human, methods of treating and methods of
diagnosing Moraxella infections in an animal, preferably a human,
and kits therefor.
Inventors: |
Tucker; Kenneth; (Frederick,
MD) ; Tillmann; Ulrich F.; (Mount Laurel,
NJ) |
Correspondence
Address: |
FOLEY AND LARDNER LLP;SUITE 500
3000 K STREET NW
WASHINGTON
DC
20007
US
|
Assignee: |
Emergent Product Development
Gaithersburg, Inc.
|
Family ID: |
22595761 |
Appl. No.: |
12/004502 |
Filed: |
December 21, 2007 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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10369299 |
Feb 19, 2003 |
7311917 |
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12004502 |
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09164714 |
Oct 1, 1998 |
6541616 |
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10369299 |
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Current U.S.
Class: |
424/450 ;
424/190.1; 435/252.1; 435/6.15; 435/7.32; 530/324; 530/350;
530/389.5 |
Current CPC
Class: |
C07K 2317/734 20130101;
A61K 39/00 20130101; C07K 16/1214 20130101; A61P 11/00 20180101;
C07K 14/212 20130101; A61K 2039/53 20130101; A61K 2035/11 20130101;
A61K 2039/523 20130101; A61K 38/00 20130101; A61P 11/02 20180101;
A61K 2039/522 20130101; A61P 31/04 20180101; A61P 27/16
20180101 |
Class at
Publication: |
424/450 ;
530/350; 530/324; 424/190.1; 435/252.1; 530/389.5; 435/7.32;
435/6 |
International
Class: |
A61K 9/127 20060101
A61K009/127; C07K 14/195 20060101 C07K014/195; C12N 1/20 20060101
C12N001/20; G01N 33/543 20060101 G01N033/543; C12Q 1/68 20060101
C12Q001/68; C07K 16/12 20060101 C07K016/12; A61K 39/02 20060101
A61K039/02 |
Claims
1. An isolated or substantially purified outer membrane protein
designated "OMP21" of Moraxella catarrhalis strain, said protein:
a) encoded by a nucleic acid comprising SEQ ID NO.: 6; or b)
comprising an amino acid sequence of SEQ ID NO.: 7; or c)
comprising an amino acid sequence of SEQ ID NO.: 1 and having a
molecular weight of about 16 kD to about 20 kD, as determined by
reducing SDS-PAGE using trypsin inhibitor and carbonic anhydrase,
respectively as 21.5 kD and 31 kD molecular weight standards; or a
fragment of said protein that specifically binds to an antibody
that specifically binds to OMP21 comprising an amino acid sequence
of SEQ ID NO.: 7.
2. The protein of claim 1, comprising the amino acid sequence of
SEQ ID NO.: 7.
3. The protein of claim 1, wherein the strain of Moraxella
catarrhalis is a virulent clinical isolate.
4. The protein of claim 1, wherein the OMP21 is at least 70 wt %
purified.
5. The protein of claim 1, recognizable by an antibody preparation
that specifically binds to a peptide having the amino acid sequence
of SEQ ID NO.: 1 or 7.
6. A peptide fragment of the OMP21 protein of claim 1, which
specifically binds to an antibody that specifically binds to an
OMP21 comprising an amino acid sequence of SEQ ID NO.: 1.
7. An isolated protein of claim 1, further comprising one or more
heterologous polypeptides fused to the C-terminal or N-terminal or
an internal segment of said OMP21.
8. An isolated protein of a Moraxella catarrhalis strain, having a
molecular weight of about 16 kD to about 20 kD, as determined by
reducing SDS-PAGE using trypsin inhibitor and carbonic anhydrase,
respectively as 21.5 kD and 31 kD molecular weight standards, said
protein: a) encoded by a nucleic acid sequence which hybridizes at
68 degrees C. in 0.5M NaHPO.sub.4 (pH7.2)/1 mM EDTA/7% SDS to a
nucleic acid of SEQ ID NO.: 6 or a nucleic acid sequence encoding
the amino acid sequence of SEQ ID NO.: 7; and b) eliciting an
immune response to M. catarrhalis when administered to an
animal.
9. A pharmaceutical composition, comprising an effective amount of
"OMP2" an isolated protein of Moraxella catarrhalis designated
"OMP21", said protein: a) encoded by a nucleic acid comprising SEQ
ID NO.: 6; or b) comprising an amino acid sequence of SEQ ID NO.:
7; or c) comprising an amino acid sequence of SEQ ID NO.: 1 and
having a molecular weight of about 16 kD to about 20 kD, as
determined by reducing SDS-PAGE using trypsin inhibitor and
carbonic anhydrase, respectively as 21.5 kD and 31 kD molecular
weight standards or a fragment of said protein that specifically
binds to an antibody that specifically binds to OMP21 comprising an
amino acid sequence of SEQ ID NO.: 7; and optionally one or more
adjuvants, and one or more pharmaceutically acceptable carriers or
diluents.
10. The pharmaceutical composition of claim 9, further comprising
an attenuated or inactivated cultivar of M. catarrhalis wherein the
cultivar has been genetically manipulated to have the nucleic acid
encoding OMP21 deleted and therefore non-transcribed.
11. The pharmaceutical composition of claim 9, further comprising
an attenuated or inactivated cultivar of M. catarrhalis wherein the
cultivar has been genetically manipulated to have the nucleic acid
encoding OMP21 and OMP 106 deleted and therefore
non-transcribed.
12. The pharmaceutical composition of claim 9, wherein the OMP21 is
combined with, fused to, or conjugated to one or more other
components, selected from the group consisting of lipids,
carbohydrates, proteins, an attenuated whole organism, and an
inactivated whole organism.
13. The pharmaceutical composition of claim 12, wherein the lipid
is a phospholipid.
14. The pharmaceutical composition of claim 12, wherein the
carbohydrate is a lipopolysaccharide.
15. The pharmaceutical composition of claim 12, wherein the whole
organism is selected from the group consisting of Moraxella,
Neisseria, Pseudomonas, Streptococcus, and Haemophilus.
16. The pharmaceutical composition of claim 12, wherein OMP21 the
component is combined with an other component, and wherein the
other component is a protein or a carbohydrate from Moraxella,
Neisseria, Pseudomonas, Streptococcus, or Haemophilus.
17. The pharmaceutical composition of claim 12, wherein the other
component is OMP106.
18. The pharmaceutical composition of claim 9, formulated as a
microparticle, capsule or liposome preparation.
19. An immunogenic composition, comprising an effective amount of
an isolated OMP21 protein of Moraxella catarrhalis, said protein:
a) encoded by a nucleic acid comprising SEQ ID NO.: 6; or b)
comprising an amino acid sequence of SEQ ID NO.: 7; or c)
comprising an amino acid sequence of SEQ ID NO.: 1 and having a
molecular weight of about 16 kD to about 20 kD, as determined by
reducing SDS-PAGE using trypsin inhibitor and carbonic anhydrase,
respectively as 21.5 kD and 31 kD molecular weight standards or a
fragment of said protein that specifically binds to an antibody
that specifically binds to OMP21 comprising an amino acid sequence
of SEQ ID NO.: 1; and one or more adjuvants, and optionally one or
more pharmaceutically acceptable carriers or diluents, wherein said
immunogenic composition produces an immune response when
administered to a host.
20. The immunogenic composition of claim 19, further comprising an
attenuated or inactivated cultivar of M. catarrhalis wherein the
cultivar has been genetically manipulated to have the nucleic acid
encoding OMP21 deleted and therefore non-transcribed.
21. The immunogenic composition of claim 19, further comprising an
attenuated or inactivated cultivar of M. catarrhalis wherein the
cultivar has been genetically manipulated to have the nucleic acid
encoding for OMP21 and OMP 106 deleted and therefore
non-transcribed.
22. The immunogenic composition of claim 19, wherein the OMP21 is
combined with, fused to, or conjugated to one or more other
components, selected from the group consisting of lipids,
carbohydrates, proteins, an attenuated whole organism and an
inactivated whole organism.
23. The immunogenic composition of claim 22, wherein the whole
organism is selected from the group consisting of Moraxella,
Neisseria, Pseudomonas, Streptococcus, and Haemophilus.
24. The immunogenic composition of claim 19, wherein the OMP21 is
combined with an other component and wherein the other component is
a protein or a carbohydrate from Moraxella, Neisseria, Pseudomonas,
Streptococcus, or Haemophilus.
25. The immunogenic composition of claim 22, wherein the other
component is OMP106.
26. The immunogenic composition of claim 19, formulated as a
microparticle, capsule, or liposome preparation.
27. A method of producing an immune response in an animal
comprising administering to said animal an effective amount of the
pharmaceutical composition of claim 9 or the immunogenic
composition of claim 19.
28. A method of preventing, treating or ameliorating a disorder
related to M. catarrhalis in an animal in need of such treatment
comprising administering an effective amount of the pharmaceutical
composition of claim 9 or the immunogenic composition of claim
19.
29. An attenuated or inactivated cultivar of M. catarrhalis wherein
the cultivar has been genetically manipulated to have the nucleic
acid encoding the OMP21 protein of claim 1 deleted and therefore
non-transcribed.
30. The attenuated or inactivated cultivar of claim 29, wherein the
cultivar is non-adherent.
31. An attenuated or inactivated cultivar of M. catarrhalis wherein
the cultivar has been genetically manipulated to have the nucleic
acid encoding the OMP21 protein of claim 1 and OMP106 deleted and
therefore non-transcribed.
32. Antisera raised against the pharmaceutical composition of claim
9 or the immunogenic composition of claim 19.
33. An isolated antibody obtainable from the antisera of claim 32
that specifically binds one or more of the components present in
the pharmaceutical composition or immunogenic composition.
34. An isolated antibody that specifically binds the OMP21 protein
of claim 1.
35. The isolated antibody of claim 31, which is a cytotoxic
antibody that mediates complement killing of Moraxella
catarrhalis.
36. The isolated antibody of claim 34, which is a cytotoxic
antibody that mediates complement killing of Moraxella
catarrhalis.
37. A method for detecting anti-M. catarrhalis antibodies in a test
sample comprising the steps of: a) contacting a test sample with
the pharmaceutical composition of claim 9 or the immunogenic
composition of claim 19 to form, in the presence of anti-M.
catarrhalis antibodies, M. catarrhalis antigen: anti-M. catarrhalis
antibody immunocomplexes, and b) detecting any said immunocomplexes
formed during step a) as an indication of the presence of said
anti-M. catarrhalis antibodies in the test sample.
38. The method of claim 37, further comprising c) measuring the
amount of immunocomplexes formed.
39. A diagnostic kit for detecting antibodies to M. catarrhalis,
said kit comprising the isolated OMP21 of claim 1, the
pharmaceutical composition of claim 9 or immunogenic composition of
claim 19, a container means for contacting said composition with a
test sample suspected of having antibodies to M. catarrhalis and a
reagent means for detecting M. catarrhalis antigen: anti-M.
catarrhalis antibody immunocomplexes formed between said
composition and said antibodies.
40. A method for detecting the presence of M. catarrhalis in a test
sample comprising the steps of: a) contacting a test sample with
the antibody of claim 33 or 34 for a time sufficient to allow said
antibody to bind M. catarrhalis, if present, to form M.
catarrhalis: anti-M. catarrhalis antibody immunocomplexes, and b)
detecting said immunocomplexes formed during step a) as an
indication of the presence of said M. catarrhalis in the test
sample.
41. The method of claim 40, further comprising c) measuring the
amount of immunocomplexes formed.
42. A diagnostic kit for detecting the presence of M. catarrhalis,
said kit comprising the antibody of claim 32, a container means for
connecting said antibody with a test sample suspected of having
said M. catarrhalis and a reagent means for measuring M.
catarrhalis: anti-M. catarrhalis antibody immunocomplexes formed
between said antibodies and said M. catarrhalis.
43. A method for determining the presence of nucleic acid encoding
OMP21 in a sample, comprising the steps of: a) contacting a sample
with a nucleic acid molecule encoding the OMP21 of claim 1 or a
complement of said nucleic acid molecule to produce duplexes
comprising the nucleic acid molecule and any said nucleic acid
molecule encoding the OMP21 in the sample and specifically
hybridizable therewith; and b) detecting duplexes produced.
44. A diagnostic kit for determining the presence of nucleic acid
encoding OMP21 in a sample, comprising: a) the nucleic acid
molecule encoding the OMP21 of claim 1 or a complement of said
nucleic acid molecule; b) a means for contacting the nucleic acid
with a sample to produce duplexes comprising the nucleic acid
molecule and any said nucleic acid molecule encoding the OMP21 in
the sample and specifically hybridizable therewith; and c) means
for detecting duplexes produced.
Description
1. FIELD OF THE INVENTION
[0001] The present invention generally relates to an isolated or
substantially purified protein obtainable from the outer membranes
of M. catarrhalis, called "OMP21" (defined below in Section 3). The
invention also encompasses the amino acid sequence thereof, and
antibodies, including cytotoxic antibodies, that specifically bind
OMP21. The invention further encompasses pharmaceutical
compositions, including prophylactic or therapeutic compositions,
and which may be immunogenic compositions, including vaccines. The
invention additionally provides methods of preventing, treating or
ameliorating disorders in mammals related to M. catarrhalis
infections and for inducing immune responses to M. catarrhalis. The
invention further provides isolated nucleotide sequences encoding
the OMP21, homologous and complementary sequences thereto, vectors
having said sequences, host cells containing said vectors, and
prophylactic or therapeutic compositions, which may be immunogenic
compositions, including vaccines comprising same. Diagnostic
methods and kits are also included.
2. BACKGROUND OF THE INVENTION
[0002] Moraxella catarrhalis, also known as Moraxella (Branhamella)
catarrhalis or Branhamella catarrhalis and formerly known as
Neisseria catarrhalis or Micrococcus catarrhalis, is a
gram-negative bacterium frequently found in the respiratory tract
of humans. M. catarrhalis, originally thought to be a harmless
commensal organism, is now recognized as an important pathogen in
upper and lower respiratory tract infections in humans. In humans,
M. catarrhalis causes serious lower respiratory tract infections in
adults with chronic lung disease, systemic infections in
immunocompromised patients, and otitis media and sinusitis in
infants and children (Helminen et al., 1993, Infect. Immun.
61:2003-2010; Catlin, B. W., 1990, Clin. Microbiol. Rev. 3:293-320;
and references cited therein). The outer surface components of
Moraxella catarrhalis have been studied in attempts to understand
the pathogenic process of M. catarrhalis infections and to develop
useful therapeutic treatments and prophylactic measures against
such infections. The outer membrane proteins (OMPs) in particular
have received considerable attention as possible virulence factors
and as potential vaccine antigens. M. catarrhalis has over 20
different OMPs with 6 to 8 of these, OMPs A to H, as the
predominate species (Murphy and Loeb, 1989, Microbial Pathogen.
6:159-174). The molecular weights of OMPs A to H range from 98 to
21 kD, respectively (Bartos and Murphy, 1988, J. Infect. Dis.
158:761-765; Helminen et al., 1993, Infect. Immun. 61:2003-2010;
Murphy et al, 1993, Molecul. Microbiol. 10:87-97; and Sarwar et al,
1992, Infect. Immun. 60:804-809). Comparisons of protein profiles
by sodium dodecylsulfate polyacrylamide gel electrophoresis
(SDS-PAGE) of outer membrane preparations from 50 M. catarrhalis
strains show nearly homogeneous patterns of OMPs A to H (Bartos and
Murphy, 1988, J. Infect. Dis. 158:761-765).
[0003] In intact bacterium or bacterially-derived outer membrane
vesicles, several of the above-identified OMPs present
surface-exposed epitopes that elicit the production of antibodies
that bind the OMPs. These antigenic OMPs include OMP E and OMP G
(Murphy and Bartos, 1989, Infect. Immun. 57:2938-2941); OMP C/D
(Sarwar et al., 1992, Infect. Immun. 60:804-809); CopB, an 80 kD
OMP, (Helminen et al., 1993, Infect. Immun. 61:2003-2010); and UspA
(Helminen et al., 1994, J. Infect. Dis. 170:867-872).
[0004] The therapeutic potential of antibodies to surface-exposed
epitopes of outer-membrane proteins of M. catarrhalis is generally
examined by the cytotoxic (bactericidal) activity, because there is
no animal model of disease. The only natural host for disease
caused by Moraxella is human. However, others have studied the role
of antibodies in an animal model of Moraxella lung clearance. The
model involved direct bolus inoculation of lungs of BALB/c VAF/Plus
mice with a controlled number of M. catarrhalis cells and
subsequent examination of the rate of pulmonary clearance of the
bacteria (Unhanand et al., 1992, J. Infect. Dis. 165:644-650).
Different clinical isolates of the M. catarrhalis exhibited
different rates of clearance, all of which are relatively rapid,
that correlated with the level of granulocyte recruitment into the
infection site. Passive immunization with a monoclonal antibody
directed to a surface-exposed epitope of CopB and UspA increased
the rate of pulmonary clearance of M. catarrhalis (Helminen et al.,
1993, Infect. Immun. 61:2003-2010; Helminen et al., 1994, J.
Infect. Dis. 170:867-872). There remains a need for compositions
and methods for diagnosis of, as well as, prophylactic and
therapeutic treatments for infections caused by M. catarrhalis.
[0005] The adherence of bacterial pathogens to a host cell surface
promotes colonization and initiates pathogenesis. See, E. H.
Beachey, 1981, J. Infect. Dis. 143:325-345. Gram-negative bacteria
typically express surface lectins that bind to specific
oligosaccharides of glycoproteins and/or glycolipids on the host
cell surface. Such lectins are often associated with pili or
fimbriae. Bacterial adherence can also occur by non-specific
binding resulting from hydrophobic and/or charge interaction with
the host cell surface.
[0006] The mechanism of M. catarrhalis adherence to cells of the
respiratory tract remains poorly understood. The organism adheres
to cultured human nasopharyngeal epithelial cells. Another study
suggests that fimbriae may have a role in the adherence to such
cells as fimbriae denaturation or treatment with anti-fimbriae
antibodies reduced adherence by fimbriated strains. Fimbriae
mediated binding, however, cannot be the sole basis of this
adherence as the most highly adhering strain, among the several
examined, was a non-fimbriated strain. Thus, other unidentified
components are involved in the bacteria's adherence.
3. SUMMARY OF THE INVENTION
[0007] An object of the present invention is to provide an isolated
or substantially purified OMP21 protein of a M. catarrhalis strain,
wherein the apparent molecular weight is about 16 kD to about 20
kD, as predicted from the deduced amino acid sequence or determined
by sodium dodecylsulfate polyacrylamide gel electrophoresis
("SDS-PAGE"). The term "OMP21" as used herein and in the claims is
intended to globally encompass: all forms of the protein having
molecular weight of 16 kd to 20 kd, including the native, wild-type
OMP21 protein obtainable from M. catarrhalis, and "OMP21-derived
polypeptides", as defined in Section 3.1 herein. Preferably, OMP21
has the sequence of any of SEQ ID Nos.: 1 or 7 or sequences
substantially homologous thereto. More preferably, OMP21 is an
outer membrane protein. Still more preferably, OMP21 has a
nasopharyngeal binding domain.
[0008] It is intended that OMP21 obtainable from any commercially
available strains and clinical isolates of Moraxella catarrhalis is
included in this invention, however preferred is OMP21 obtainable
from virulent clinical isolates. The OMP21 is at least 70 wt %
pure, preferably at least about 90 wt % pure, and may be in the
form of an aqueous solution thereof.
[0009] Another object of the present invention is to provide an
isolated nucleic acid molecule encoding OMP21. Preferred is the
nucleic acid sequence wherein the encoded OMP21 comprises the amino
acid sequence of any of SEQ ID NOs.: 1 or 7 or sequences
substantially homologous thereto. Also included is an isolated
nucleic acid molecule comprising a sequence of any of SEQ ID NOs:
2-6 and 8-20, a complementary sequence thereof, sequences
substantially homologous thereto, and any fragment thereof; a DNA
sequence encoding a deduced amino acid sequence of any of SEQ ID
Nos.:1 or 7, the complimentary sequence thereto, sequences
substantially homologous thereto, and any fragment thereof; and a
nucleic acid sequence which hybridizes to any one of the sequences
described above. The nucleic acid that hybridizes under stringent
conditions preferably has a sequence identity of about 70% with any
of the sequences mentioned above, more preferably about 90%.
[0010] Another object, of the invention is to provide a recombinant
expression vector adapted for transformation of a host or for
delivery of a sequence encoding OMP21 to a host, comprising the
nucleic acid molecule of SEQ ID NO: 6, a complementary sequence
thereof, sequences substantially homologous thereto, and any
fragment thereof. Preferably, the recombinant expression vector is
adapted for transformation of a host and comprises an expression
means operatively coupled to the nucleic acid molecule for
expression by the host of said OMP21. More preferred is the
expression vector wherein the expression means includes a nucleic
acid portion encoding a leader sequence for secretion or
purification from the host of OMP21.
[0011] A further aspect of the invention includes a transformed
host cell comprising an expression vector described above and OMP21
producible by the transformed host cell.
[0012] The invention further encompasses attenuated and/or
inactivated cultivars of M. catarrhalis wherein the cultivar has
been genetically manipulated to have the gene for OMP21
"knocked-out" and therefore non-transcribed (a "deletion-mutant"),
so that the adherence by the organism is reduced. Also encompassed
in this invention are cultivars of M. catarrhalis having a double
deletion of OMP21 and OMP106, as described in PCT publication WO
97/41731, which is incorporated herein by reference in its
entirety, so that the organism is non-adherent.
[0013] The invention further encompasses pharmaceutical
compositions, including prophylactic and therapeutic compositions,
and which may be immunogenic compositions including vaccines,
wherein said immunogenic composition produces an immune response
when administered to a host, comprising at least one component
selected from the following group: [0014] a) OMP21; [0015] b) a
nucleic acid molecule or a fragment or compliment thereof, encoding
OMP21; [0016] c) a nucleic acid molecule having the sequence of SEQ
ID NO:6, the complimentary sequence thereto, a nucleic acid
sequence which hybridizes under stringent conditions thereto, or
fragments thereof; [0017] d) OMP21, obtainable from a transformed
host comprising an expression vector comprising a nucleic acid
molecule as defined in b) or c) and expression means operatively
coupled to the nucleic acid molecule for expression by the host of
said OMP21; [0018] e) a recombinant vector comprising a nucleic
acid or fragment or analog thereof, encoding OMP21; and [0019] f) a
transformed cell comprising the vector of e), optionally one or
more adjuvants, and optionally a pharmaceutically acceptable
carrier or diluent.
[0020] The invention further encompasses pharmaceutical
compositions, including prophylactic and therapeutic compositions,
and which may be immunogenic compositions including vaccines,
comprising an attenuated and/or inactivated M. catarrhalis cultivar
provided herein, optionally an adjuvant, and optionally a
pharmaceutically acceptable carrier or diluent.
[0021] The invention further encompasses the pharmaceutical
compositions described above, optionally in combination with, fused
to, or conjugated to another component, which may be an immunogen,
and may include but is not limited to: a lipid, a phospholipid, a
carbohydrate including a lipopolysaccharide, another protein, and
an attenuated or inactivated whole organism, as provided herein or
known to those skilled in the art, including but not limited to
those described in Section 2 above. Preferred optional components
include any Moraxella, Neisseria, Pseudomonas, Streptococcus, or
Haemophilus attenuated or inactivated whole organism, or a protein
or a carbohydrate therefrom. Preferred immunogenic compositions,
including vaccines, comprise OMP21 in combination with OMP106 and
one or more adjuvants.
[0022] Also included are methods for producing an immune response
in an animal comprising administering to said animal an effective
amount of an immunogenic composition described above.
[0023] Another aspect of the invention is directed to the antisera
raised against any of the immunogenic compositions described above,
and the antibodies present in the antisera that specifically bind
the immunogens present in the immunogenic composition, including
OMP21 or nucleic acid encoding same, and other immunogenic
components.
[0024] The invention also includes diagnostic reagents which may
include any of the above mentioned aspects, such as the isolated
OMP21, the nucleic acid molecule encoding OMP21, the immunogenic
composition, the antisera, the antibodies, the vector comprising
the nucleic acid, and the transformed cell comprising the
vector.
[0025] Methods and diagnostic kits for detecting OMP21, M.
catarrhalis, anti-OMP21 antibodies or anti-M. catarrhalis
antibodies in a test sample are also included, wherein the methods
comprise the steps of: [0026] a) contacting a test sample with an
antigenic or immunogenic composition of the present invention or
antibodies thereto to form antigen:antibody immunocomplexes, and
further, [0027] b) detecting any immunocomplexes formed during step
a) as an indication of the presence of said antigen or antibodies
in a test sample. The methods may further comprise quantitating any
said immunocomplexes formed. The diagnostic kits for detecting
OMP21, M. catarrhalis, or antibodies thereto, comprise the
antibodies and/or the antigenic or immunogenic composition of the
present invention, a container means for contacting said antibodies
or antigenic or immunogenic composition with a test sample
suspected of having said antibodies and reagent means for measuring
antigen:antibody immunocomplexes formed between said antigenic or
immunogenic composition and said antibodies.
[0028] A further aspect of the present invention provides methods
for determining the presence of nucleic acids encoding OMP21 in a
sample, comprising the steps of: [0029] a) contacting a sample with
the nucleic acid molecule provided herein to produce duplexes
comprising the nucleic acid molecule and any nucleic acid molecule
encoding the OMP21 in the sample and specifically hybridizable
therewith; and [0030] b) determining the production of
duplexes.
[0031] The present invention also provides a diagnostic kit and
reagents therefor, for determining the presence of nucleic acid
encoding OMP21 in a sample, comprising: [0032] a) the nucleic acid
molecule as provided herein; [0033] b) means for contacting the
nucleic acid with the sample to produce duplexes comprising the
nucleic acid molecule and any nucleic acid molecule encoding the
OMP21 in the sample and specifically hybridizable therewith; and
[0034] b) means for determining the production of duplexes.
[0035] Also included in this invention are methods of preventing,
treating or ameliorating disorders related to M. catarrhalis in an
animal, preferably a human, in need of such treatment comprising
administering an effective amount of a pharmaceutical composition
provided herein. Preferred disorders include a M. catarrhalis
bacterial infection, otitis media, respiratory infections,
sinusitis and pneumonia. Preferred vaccines and pharmaceutical
compositions include those formulated for in vivo administration to
an animal, preferably a human, to confer protection against
disease, or treatment therefor, caused by a strain of M.
catarrhalis. Also preferred are compositions formulated as a
microparticle, capsule, or liposome preparation.
3.1. Definitions and Abbreviations
[0036] anti-OMP21=anti-OMP21 polypeptide antibody or antiserum
[0037] ATCC=American Type Culture Collection [0038] Blebs=naturally
occurring outer membrane vesicles of M. catarrhalis [0039]
immunogen & immnuogenic=capable of provoking a cellular or
humoral immune response [0040] kD=kilodaltons [0041] M.
catarrhalis=M.c.; Moraxella catarrhalis; Moraxella (Branhamella)
catarrhalis; Branhamella catarrhalis; Neisseria catarrhalis; or
Micrococcus catarrhalis [0042] OG=n-octyl .beta.-D-glucopyranoside
or octyl glucoside [0043] OMP21="wild-type" protein obtainable from
outer membranes of Moraxella catarrhalis, having a molecular weight
of about 16 kD to about 20 kD, as predicted from the deduced amino
acid sequence or as determined by SDS-PAGE; and OMP21-derived
polypeptides obtainable from any source by any means including
chemical synthesis and recombinant synthesis [0044] OMP21-derived
polypeptide=any variant or analog of wild-type protein obtainable
from outer membranes of Moraxella catarrhalis, having a molecular
weight or about 16 kD to about 20 kD ("wild-type-OMP21") containing
one or more amino acid deletions, insertions or substitutions; any
fragment of wild-type-OMP21 or any variant or analog thereof; any
chimeric protein comprising a heterologous polypeptide fused to the
C-terminal or N-terminal or internal segment of a whole or a
portion of wild-type-OMP21 or any fragment, variant or analog
thereof [0045] OMP=outer membrane protein [0046] OMPs=outer
membrane proteins [0047] PBS=phosphate buffered saline [0048]
PA=polyacrylamide gel [0049] polypeptide=a peptide of any length,
preferably one having eight or more amino acid residues [0050]
SDS=sodium dodecylsulfate [0051] SDS-PAGE=sodium dodecylsulfate
polyacrylamide gel electrophoresis
[0052] Nucleotide or nucleic acid sequences defined herein are
represented by one-letter symbols for the bases as follows:
A (adenine) C (cytosine) G (guanine) T (thymine) U (uracil)
M (A or C)
R (A or G)
W (A or T/U)
S(C or G)
Y (C or T/U)
K (G or T/U)
V (A or C or G; not T/U)
H (A or C or T/U; not G)
D (A or G or T/U; not C)
B (C or G or T/U; not A)
[0053] N (A or C or G or T/U) or (unknown)
[0054] Peptide and polypeptide sequences defined herein are
represented by one-letter or three-letter symbols for amino acid
residues as follows:
TABLE-US-00001 1 letter 3 letter amino acid A Ala (alanine) R Arg
(arginine) N Asn (asparagine) D Asp (aspartic acid) C Cys
(cysteine) Q Gln (glutamine) E Glu (glutamic acid) G Gly (glycine)
H His (histidine) I Ile (isoleucine) L Leu (leucine) K Lys (lysine)
M Met (methionine) F Phe (phenylalanine) P Pro (proline) S Ser
(serine) T Thr (threonine) W Trp (tryptophan) Y Tyr (tyrosine) V
Val (valine) X Xaa (unknown)
[0055] The present invention may be more fully understood by
reference to the following detailed description of the invention,
non-limiting examples of specific embodiments of the invention and
the appended figures.
4. BRIEF DESCRIPTION OF THE FIGURES
[0056] FIG. 1: Denaturing PAGE comparison of outer membrane protein
profiles of M. catarrhalis blebs or octyl glucoside (OG) extracts
of whole M. catarrhalis cells. The numbers over the lanes (81176,
23246, 25238 and 49143) refer to the ATCC strain designations. A
prestained SDS-PAGE standard (BioRad catalog #161-0305) was used as
molecular weight markers. The standard consisted of the following
polypeptides with their approximate molecular weights noted in
parenthesis: rabbit muscle phosphorylase B (106 kD); bovine serum
albumin (80 kD); hen egg white ovalbumin (49.5 kD) bovine carbonic
anhydrase (32.5 kD), soybean trypsin inhibitor (27.5 kD); hen egg
white lysozyme (18.5 kD). The positions of the molecular weight
markers in the gel are noted on the left side of the drawing by
arrows with the molecular weights (kD) of some of the markers above
the arrows.
[0057] FIG. 2: Molecular weight estimation of OMP21 in a 4-20%
gradient denaturing polyacrylamide gel in the presence of a
reducing agent using OMP21 purified from ATCC strain 49143 that
were incubated in sample buffer at either 25.degree. C. (Lanes 1-3)
or 100.degree. C. (Lanes 4-6) prior to application to the gel.
Proteins in the gel were visualized by reductive silver staining. A
broad range SDS-PAGE standard (NOVEX, catalog #LC5677) was used as
molecular weight markers (shown on left). The standards consisted
of the following polypeptides (approximate molecular weights noted
in parenthesis): rabbit skeletal muscle myosin (200 kD); E. coli
.beta.-galactosidase (116 kD); phosphorylase B (97.4 kD); bovine
serum albumin (66.2 kD); glutamic dehydrogenase (55.4 kD); lactate
dehydrogenase (36.5 kD); carbonic anhydrase (31 kD); trypsin
inhibitor (21.5 kD); lysozyme (14.4 kD); and aprotinin (6 kD). The
positions of the molecular weight markers in the gel are noted on
the left side of the figure by lines with the molecular weights
(kD) of the markers above the lines.
[0058] FIG. 3: Determined nucleic acid sequence of OMP21 from M.
catarrhalis strain 49143.
[0059] FIG. 4: Deduced amino acid sequence of OMP21 from M.
catarrhalis strain 49143.
[0060] FIG. 5: RFLP Southern blot analysis of HindIII restriction
endonuclease digests of M. catarrhalis chromosomal DNA as described
in the Example in Section 11. The first lane contains size markers
(M); the other lanes (B, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 17, 20)
contain the hybridizing bands to the digests that all have an
approximate size of 1.8 kb.
[0061] FIG. 6: Southern blot analysis of PstI restriction
endonuclease digests of M. catarrhalis wild-type and OMP21
deletion-mutant (knock-out) DNA as described in the Example in
Section 10.5. Using the probe described, a .sup..about.8 kb DNA
fragment is detected in PstI digests of wild-type M. catarrhalis
DNA (MC2926). The same probe detects a .sup..about.4.5 kb DNA
fragment in digests of the knock-outs (KO1, KO4). Size markers are
indicated on the left.
[0062] FIGS. 7A and 7B: Western Blots of protein extracts of
several M. catarrhalis strains (shown in both 7A (top panel) and 7B
(bottom panel)) using a rabbit antiserum to OMP21 (the location of
the OMP21 polypeptide is indicated by the arrow) as the probe. The
prestained molecular weight markers (NOVEX, catalog #LC5725)
consisted of the following polypeptides (approximate molecular
weights noted in parenthesis): myosin (250 kD); phosphorylase B
(148 kD); glutamic dehydrogenase (60 kD); carbonic anhydrase (42
kD); myoglobin (30 kD); lysozyme (17 kD); aprotinin (6 kD); and
insulin (4 kD).
[0063] FIG. 8: Western Blot of protein extracts of OMP21-deletion
mutants of M. catarrhalis using a rabbit antiserum to OMP21 as the
probe. Octyl glucoside extracts of the parent strain (Lane 2) and
OMP21 deletion mutant M. catarrhalis strains (Lanes 3-5). The
transfer and Western blot procedures and molecular weight markers
(Lane 1) used were identical to those used to obtain the blots
shown in FIGS. 7A and B.
[0064] FIG. 9: Map of OMP21 and OMP21 deletion mutants. The
organization of the omp 21 locus in the wild-type strain compared
to the structure imposed on the locus after the gene-targeting
construct has been inserted by homologous recombination is shown.
The map also shows restriction endonuclease cleavage sites as well
as DNA fragments of 80 bp and 543 bp described in the Examples
below.
5. DETAILED DESCRIPTION OF THE INVENTION
5.1. OMP21 Polypeptide
[0065] OMP21 polypeptide of the invention is the outer membrane
protein of a M. catarrhalis strain or cultivar that has an apparent
molecular weight in SDS-PAGE of about 16 kD to about 20 kD.
According to the invention, an outer membrane protein of M.
catarrhalis is a polypeptide that is present in M. catarrhalis
blebs, or that can be extracted from M. catarrhalis blebs or intact
cells by a detergent, such as but not limited to any n-octyl
.beta.-D-glucopyranoside (OG), EmpigenBB.TM.
(N-dodecyl-N,N-dimethyl-glycine, CalBiochem) and sarkosyl, in
buffer solution at room temperature. See Murphy and Loeb, 1989,
Microbial Pathogenesis 6:159-174, for a discussion of M.
catarrhalis blebs, which are naturally occurring vesicles
consisting of the outer membrane of M. catarrhalis.
[0066] OMP21 polypeptide may also be identified as the polypeptide
in extract of M. catarrhalis blebs or intact cells that has an
apparent molecular weight of about 16 kD to about 20 kD as
determined by denaturing gel electrophoresis in PA with SDS, using
formulations as described in Harlow and Lane (Antibodies: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., Appendix I, 1988). In a particular embodiment, OMP21
polypeptide in the detergent extract of M. catarrhalis strain ATCC
49143 has an apparent molecular weight of about 16-20 kD. Heat
treatment of the detergent extract at 100.degree. C. for 5 minutes
does not modify the apparent molecular weight of OMP21 polypeptide
as determined by SDS-PAGE in 4-20% gradient PAG with a reducing
agent (.beta.-mercaptoethanol) using formulations described in
Harlow and Lane, supra. See FIG. 2.
[0067] In particular embodiments, the OMP21 polypeptide is that
obtainable from any of M. catarrhalis strains including, but not
limited to, ATCC 49143, ATCC 25238, ATCC 25240, ATCC 43617, ATCC
43618, ATCC 43627 and ATCC 43628. The preferred source of OMP21
polypeptide is ATCC 49143.
[0068] In a particular embodiment, OMP21 comprises, preferably at
the amino-terminal, the amino acid sequence
AISYGNSADAQPYVGAKIGQVDAKQINGKNTAYGIYAGYN (SEQ ID NO:1) or a
sequence substantially homologous thereto. In another particular
embodiment, OMP21 comprises the deduced amino acid sequence (SEQ ID
NO:7) or a sequence substantially homologous thereto.
[0069] As used herein, a "substantially homologous" sequence is at
least 80%, preferably greater than 80%, more preferably greater
than 90% identical to a reference sequence of identical size or
when compared to a reference sequence when the alignment or
comparison is conducted by a computer homology program or search
algorithm known in the art. By way of example and not limitation,
useful computer homology programs include the following: Basic
Local Alignment Search Tool (BLAST) (www.ncbi.nlm.nih.gov)
(Altschul et al., 1990, J. of Molec. Biol., 215:403-410, "The BLAST
Algorithm; Altschul et al., 1997, Nuc. Acids Res. 25:3389-3402) a
heuristic search algorithm tailored to searching for sequence
similarity which ascribes significance using the statistical
methods of Karlin and Altschul 1990, Proc. Nat'l Acad. Sci. USA,
87:2264-68; 1993, Proc. Nat'l Acad. Sci. USA 90:5873-77. Five
specific BLAST programs perform the following tasks:
[0070] 1) The BLASTP program compares an amino acid query sequence
against a protein sequence database.
[0071] 2) The BLASTN program compares a nucleotide query sequence
against a nucleotide sequence database.
[0072] 3) The BLASTX program compares the six-frame conceptual
translation products of a nucleotide query sequence (both strands)
against a protein sequence database.
[0073] 4) The TBLASTN program compares a protein query sequence
against a nucleotide sequence database translated in all six
reading frames (both strands).
[0074] 5) The TBLASTX program compares the six-frame translations
of a nucleotide query sequence against the six-frame translations
of a nucleotide sequence database.
[0075] Smith-Waterman (database: European Bioinformatics Institute
wwwz.ebi.ac.uk/bic_sw/) (Smith-Waterman, 1981, J. of Molec. Biol.,
147:195-197) is a mathematically rigorous algorithm for sequence
alignments.
[0076] FASTA (see Pearson et al., 1988, Proc. Nat'l Acad. Sci. USA,
85:2444-2448) is a heuristic approximation to the Smith-Waterman
algorithm. For a general discussion of the procedure and benefits
of the BLAST, Smith-Waterman and FASTA algorithms see Nicholas et
al., 1998, "A Tutorial on Searching Sequence Databases and Sequence
Scoring Methods" (www.psc.edu) and references cited therein.
[0077] According to various aspects of the invention, the
polypeptides of the invention are characterized by their apparent
molecular weights based on the polypeptides' migration in SDS-PAGE
relative to the migration of known molecular weight markers. While
any molecular weight standards known in the art may be used with
the SDS-PAGE, preferred molecular weight markers comprise carbonic
anhydrase, trypsin inhibitor and lysozyme. One skilled in the art
will appreciate that the polypeptides of the invention may migrate
differently in different types of gel systems (e.g., different
buffers; different types and concentrations of gel, crosslinker or
SDS). One skilled in the art will also appreciate that the
polypeptides may have different apparent molecular weights due to
different molecular weight markers used with the SDS-PAGE. Hence,
the molecular weight characterization of the polypeptides of the
invention is intended to be directed to cover the same polypeptides
on any SDS-PAGE systems and with any molecular weight markers which
might indicate slightly different apparent molecular weights for
the polypeptides than those disclosed here.
5.2. OMP21-Derived Polypeptides
[0078] OMP21-derived polypeptides are intended to be encompassed by
the term OMP21, and may be a fragment of the OMP21 polypeptide
having 6 or more amino acids, preferably 8 or more amino acids,
more preferably 9 or more amino acids, still more preferably 10 or
more amino acids. The intact OMP21 polypeptide may contain one or
more amino acid residues that are not necessary to its
immunogenicity. It may be the case, for example, that only the
amino acid residues forming a particular epitope of the OMP21
polypeptide are necessary for immunogenic activity. Unnecessary
amino acid sequences can be removed by techniques well known in the
art. For example, the unwanted amino acid sequences can be removed
by limited proteolytic digestion using enzymes such as trypsin,
papain, or related proteolytic enzymes or by chemical cleavage
using agents such as cyanogen bromide and followed by fractionation
of the digestion or cleavage products.
[0079] An OMP21-derived polypeptide of the invention may also be a
modified OMP21 polypeptide or fragment thereof (i.e., an OMP21
polypeptide or fragment having one or more amino acid
substitutions, insertions and/or deletions of the wild-type OMP21
sequence). Such modifications may enhance the immunogenicity of the
resultant polypeptide product or have no effect on such activity.
Modification techniques that may be used include those disclosed in
U.S. Pat. No. 4,526,716.
[0080] As an illustrative, non-limiting example, one or more amino
acid residues within the sequence can be substituted by another
amino acid of a similar polarity which acts as a functional
equivalent, resulting in a silent alteration. Substitutes for an
amino acid within the sequence may be selected from other members
of the class to which the amino acid belongs. For example, the
nonpolar (hydrophobic) amino acids include alanine, leucine,
isoleucine, valine, proline, phenylalanine, tryptophan and
methionine. The polar neutral amino acids include glycine, serine,
threonine, cysteine, tyrosine, asparagine, and glutamine. The
positively charged (basic) amino acids include arginine, lysine and
histidine. The negatively charged (acidic) amino acids include
aspartic acid and glutamic acid.
[0081] An OMP21-derived polypeptide of the invention may also be a
molecule comprising a region that is substantially homologous to
(e.g., in various embodiments, at least 60% or 70% or 80% or 90% or
95% identity over an amino acid sequence of identical size or when
compared to an aligned sequence in which the alignment is performed
by a computer homology program known in the art) or whose encoding
nucleic acid is capable of hybridizing to a coding OMP21 sequence,
under highly stringent, moderately stringent, or low or
nonstringent conditions.
[0082] By way of example and not limitation, useful computer
homology programs include the following: Basic Local Alignment
Search Tool (BLAST) (www.ncbi.nlm.nih.gov) (Altschul et al., 1990,
J. of Molec. Biol., 215:403-410, "The BLAST Algorithm; Altschul et
al., 1997, Nuc. Acids Res. 25:3389-3402) a heuristic search
algorithm tailored to searching for sequence similarity which
ascribes significance using the statistical methods of Karlin and
Altschul (1990, Proc. Nat'l Acad. Sci. USA, 87:2264-68; 1993, Proc.
Nat'l Acad. Sci. USA 90:5873-77). Two specific BLAST programs
perform the following tasks:
[0083] 1) The BLASTP program compares an amino acid query sequence
against a protein sequence database; and
[0084] 2) The BLASTN program compares a nucleotide query sequence
against a nucleotide sequence database; and hence are useful to
identify, respectively, substantially homologous amino acid and
nucleotide sequences.
[0085] Additional algorithms which can be useful are the
Smith-Waterman and FASTA algorithms. See supra, Section 5.1.
[0086] Included within the scope of the invention are OMP21-derived
polypeptides which are OMP21 polypeptide fragments or other
derivatives or analogs which are differentially modified during or
after translation, e.g., by glycosylation, acetylation,
phosphorylation, amidation, derivatization by known
protecting/blocking groups, proteolytic cleavage, linkage to an
antibody molecule or other cellular ligand, etc. Any of numerous
chemical modifications may be carried out by known techniques,
including but not limited to specific chemical cleavage by cyanogen
bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH.sub.4;
acetylation, formylation, oxidation, reduction; metabolic synthesis
in the presence of tunicamycin; etc.
[0087] Furthermore, if desired, nonclassical amino acids or
chemical amino acid analogs can be introduced as a substitution or
addition into the OMP21 polypeptide sequence. Nonclassical amino
acids include but are not limited to the D-isomers of the common
amino acids, .alpha.-amino isobutyric acid, 4-aminobutyric acid,
Abu, 2-amino butyric acid, .gamma.-Abu, .epsilon.-Ahx, 6-amino
hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic
acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine,
citrulline, cysteic acid, t-butylglycine, t-butylalanine,
phenylglycine, cyclohexylalanine, .beta.-alanine, fluoro-amino
acids, designer amino acids such as .beta.-methyl amino acids,
C.alpha.-methyl amino acids, N.alpha.-methyl amino acids, and amino
acid analogs in general. Furthermore, the amino acid can be D
(dextrorotary) or L (levorotary).
[0088] An OMP21-derived polypeptide may further be a chimeric
polypeptide comprising one or more heterologous polypeptides fused
to the amino-terminal or carboxyl-terminal or internal of a
complete OMP21 polypeptide or a portion of or a fragment thereof.
Useful heterologous polypeptides comprising such chimeric
polypeptide include, but are not limited to, a) pre- and/or
pro-sequences that facilitate the transport, translocation and/or
processing of the OMP21-derived polypeptide in a host cell, b)
affinity purification sequences, and c) any useful immunogenic
sequences (e.g., sequences encoding one or more epitopes of a
surface exposed protein of a microbial pathogen).
[0089] Preferably, the OMP21-derived polypeptides of the invention
are immunologically cross-reactive with wild-type-OMP21, thus being
capable of eliciting in an animal an immune response to M.
catarrhalis. More preferably, the OMP21-derived polypeptides of the
invention comprise sequences forming one or more outer surface
epitopes of the native OMP21 polypeptide of M. catarrhalis (i.e.,
the surface exposed epitopes of OMP21 polypeptide as it exists in
intact M. catarrhalis cells). Such preferred OMP21-derived
polypeptides can be identified by their ability to specifically
bind antibodies raised to intact M. catarrhalis cells (e.g.,
antibodies elicited by formaldehyde or glutaraldehyde fixed M.
catarrhalis cells; such antibodies are referred to herein as
"anti-whole cell" antibodies). For example, polypeptides or
peptides from a limited or complete protease digestion of the OMP21
polypeptide are fractionated using standard methods and tested for
their ability to bind anti-whole cell antibodies. Reactive
polypeptides comprise preferred OMP21-derived polypeptides. They
are isolated and their amino acid sequences determined by methods
known in the art.
[0090] These preferred OMP21-derived polypeptides also can be
identified by using anti-whole cell antibodies to screen bacterial
libraries expressing random fragments of M. catarrhalis genomic DNA
or cloned nucleotide sequences encoding the OMP21 polypeptide. See,
e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd
ed., Cold Spring Harbor Press, NY, Vol. 1, Chapter 12. The reactive
clones are identified and their inserts are isolated and sequenced
to determine the amino acid sequences of such preferred
OMP21-derived polypeptides.
5.3. Isolation and Purification of OMP21
[0091] The invention provides isolated OMP21. As used herein, the
term "isolated" means that the product is significantly free of
other biological materials with which it is naturally associated.
That is, for example, an isolated OMP21 is between about 70% and
94% pure OMP21 by weight. Preferably, OMP21 of the invention is
purified. As used herein, the term "purified" means that OMP21 is
substantially free of other biological material with which it is
naturally associated. That is, a purified OMP21 is at least 95%
pure OMP21 by weight, preferably at least 98% pure OMP21 by weight,
and most preferably at least 99% pure OMP21 by weight.
[0092] The OMP21 of the invention may be isolated from protein
extracts including whole cell extract, of any M. catarrhalis strain
or cultivar. Preferably, the protein extract is a detergent extract
of outer membrane vesicles (i.e., blebs) or whole cells of M.
catarrhalis including, but not limited to, any of strains ATCC
49143, ATCC 25238, ATCC 25240, ATCC 43617, ATCC 43618, ATCC 43627
and ATCC 43628. The preferred source of such extracts is ATCC
49143. Another source of the OMP21 is protein preparations from
gene expression systems expressing cloned sequences encoding OMP21
(see Section 5.8., infra).
[0093] OMP21 can be isolated and purified from the source material
using any biochemical technique and approach well known to those
skilled in the art. In one approach, M. catarrhalis outer membrane
is obtained by standard techniques and outer membrane proteins are
solubilized using a solubilizing compound such as a detergent. A
preferred solubilizing solution is one containing about 1.5% octyl
glucopyranoside w/v (OG). Another preferred solubilizing solution
is one containing about 1.0% EmpigenBB.TM.
(N-dodecyl-N,N-dimethyl-glycine, CalBiochem). OMP21 is in the
solubilized fraction. Cellular debris and insoluble material in the
extract are separated and removed preferably by centrifuging. The
polypeptides in the extract are concentrated, incubated in
SDS-containing Laemmli gel sample buffer at 100.degree. C. for 5
minutes and then fractionated by electrophoresis in a 4-20%
gradient denaturing SDS-PAGE. See Laemmli, 1970, Nature
227:680-685. The band or fraction identified as OMP21 as described
above (e.g., the silver-stained polypeptide band that is present in
the detergent extract) may then be isolated directly from the
fraction or gel slice containing the OMP21. In a preferred
embodiment, OMP21 has an apparent molecular weight of 16 to about
20 kD as determined by comparing its migration distance or rate in
a denaturing SDS-PAGE relative to those of trypsin inhibitor (21.5
kD) and lysozyme (14.4 kD).
[0094] Another method of purifying OMP21 is by affinity
chromatography using anti-OMP21 antibodies, (see Section 5.5).
Preferably, monoclonal anti-OMP21 antibodies are used. The
antibodies are covalently linked to agarose gels activated by
cyanogen bromide or succinamide esters (Affi-Gel, BioRad, Inc.) or
by other methods known to those skilled in the art. The protein
extract is loaded on the top of the gel as described above. The
contact is for a period of time and under standard reaction
conditions sufficient for OMP21 to bind to the antibody.
Preferably, the solid support is a material used in a
chromatographic column. OMP21 is then removed from the antibody,
thereby permitting the recovery OMP21 in isolated, or preferably,
purified form.
[0095] OMP21 fragments can be produced by chemical and/or enzymatic
cleavage or degradation of isolated or purified OMP21. OMP21 can
also be chemically synthesized based on the known amino acid
sequence of OMP21 and, in the case of a chimeric polypeptide, those
of the heterologous polypeptide by methods well known in the art.
See, for example, Creighton, 1983, Proteins: Structures and
Molecular Principles, W.H. Freeman and Co., NY.
[0096] OMP21 can also be produced in a gene expression system
expressing a recombinant nucleotide construct comprising sequences
encoding OMP21. The nucleotide sequences encoding polypeptides of
the invention may be synthesized, and/or cloned, and expressed
according to techniques well known to those skilled in the art.
See, for example, Sambrook, et al., 1989, Molecular Cloning, A
Laboratory Manual, Vols. 1-3, Cold Spring Harbor Press, NY, Chapter
9.
[0097] OMP21 can be fractionated and purified by the application of
standard protein purification techniques, modified and applied in
accordance with the discoveries and teachings described herein. In
particular, preferred OMP21 fragments, those that form an outer
surface epitope of the native OMP21, may be isolated and purified
according to the affinity procedures disclosed above for the
isolation and purification of OMP21 (e.g., affinity purification
using anti-OMP21 antibodies).
[0098] If desirable, the polypeptides of the invention may be
further purified using standard protein or peptide purification
techniques including but are not limited to electrophoresis,
centrifugation, gel filtration, precipitation, dialysis,
chromatography (including ion exchange chromatography, affinity
chromatography, immunoadsorbent affinity chromatography,
reverse-phase high performance liquid chromatography, and gel
permeation high performance liquid chromatography), isoelectric
focusing, and variations and combinations thereof.
[0099] One or more of these techniques may be employed sequentially
in a procedure designed to separate molecules according to their
physical or chemical characteristics. These characteristics include
the hydrophobicity, charge, binding capability, and molecular
weight of the protein. The various fractions of materials obtained
after each technique are tested for their molecular weight or their
abilities to bind anti-OMP21 antibodies. Those fractions showing
such activity are then subjected to the next technique in the
sequential procedure, and the new fractions are tested again. The
process is repeated until only one fraction having the above
described characteristics remains and that fraction produces only a
single band or entity when subjected to polyacrylamide gel
electrophoresis or chromatography.
5.4. OMP21 Immunogens and Anti-OMP21 Antibodies
[0100] As used herein and in the claims, "antibodies" of the
invention may be obtained by any conventional methods known to
those skilled in the art, such as but not limited to the methods
described in Antibodies A Laboratory Manual (E. Harlow, D. Lane,
Cold Spring Harbor Laboratory Press, 1989) which is incorporated
herein by reference in its entirety. The term "antibodies" is
intended to include all forms, such as but not limited to
polyclonal, monoclonal, purified IgG, IgA, IgM and fragments
thereof.
[0101] The present invention provides antibodies that specifically
bind OMP21. For the production of such antibodies, an
immunogenically effective amount of a composition comprising at
least one isolated or preferably, purified component selected from
the following group: [0102] a) OMP21; [0103] b) a nucleic acid
molecule or a fragment or compliment thereof, encoding OMP21;
[0104] c) a nucleic acid molecule having the sequence of SEQ ID NO:
6, the complimentary sequence thereto, a nucleic acid sequence
which hybridizes under high stringency conditions thereto, or
fragments thereof; [0105] d) OMP21, obtainable from a transformed
host comprising an expression vector comprising a nucleic acid
molecule as defined in b) or c) and expression means operatively
coupled to the nucleic acid molecule for expression by the host of
said OMP21; [0106] e) a recombinant vector comprising a nucleic
acid or fragment or analog thereof, encoding OMP21; [0107] f) a
transformed cell comprising the vector of e), optionally one or
more adjuvants, and optionally a pharmaceutically acceptable
carrier or diluent therefor are administered to an animal.
[0108] The above mentioned compositions may further include
optionally in combination with, fused to, or conjugated to another
component, which may be an immunogen, including a lipid, a
phospholipid, a carbohydrate, including a lipopolysaccharide,
another protein, and an attenuated or inactivated whole organisms,
as provided herein or known to those skilled in the art, including
but not limited to those described in Section 2 above. Preferred
optional components include any Moraxella, Neisseria or Haemophilus
protein. Preferred immunogenic compositions, including vaccines,
comprise isolated OMP21 in combination with OMP106 and one or more
adjuvants.
[0109] In an embodiment, the OMP21 is separated from other outer
membrane proteins present in the detergent extract of outer
membrane of M. catarrhalis cells or blebs using SDS-PAGE (see
Section 5.2. above) and the gel slice containing OMP21 is used as
the immunogen and injected into a rabbit to produce antisera
containing polyclonal OMP21-antibodies. The same immunogen can be
used to immunize mice and guinea pigs for the production of
polyclonal antibodies or mice for the production of hybridoma lines
that produce monoclonal anti-OMP21 antibodies. In particular
embodiments, a PA slice containing isolated or purified OMP21 from
any of strains ATCC 49143, ATCC 25238, ATCC 25240, ATCC 43617, ATCC
43618, ATCC 43627 and ATCC 43628 is used as the immunogen. In
preferred embodiments, a PA slice containing isolated or purified
OMP21 from strain ATCC 49143 is used as the immunogen.
[0110] In other embodiments, a peptide fragment of OMP21 is used as
an immunogen. Preferably, a peptide fragment of purified OMP21 or a
chemically synthesized peptide fragment of OMP21 is used. The
peptides may be produced by protease digestion, chemical cleavage
of isolated or purified OMP21 or chemical synthesis and then may be
isolated or purified. Such isolated or purified peptides can be
used directly as immunogens. In particular embodiments, useful
peptide fragments include but are not limited to those having the
sequence AISYGNSADAQPYVGAKIGQVDAKQINGKNTAYGIYAGYN (SEQ ID NO:1) or
any portion thereof that is 6 or more amino acids in length. In an
another embodiment, the peptide has the sequence as shown in FIG. 3
(SEQ ID NO: 7).
[0111] Useful immunogens may also comprise OMP21 conjugated to a
carrier molecule, preferably a carrier protein. Carrier proteins
may be any commonly used in immunology, include, but are not
limited to, bovine serum albumin (BSA), chicken albumin, keyhole
limpet hemocyanin (KLH) and the like. For a discussion of hapten
protein conjugates, see, for example, Hartlow, et al., Antibodies:
A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y., 1988, or a standard immunology textbook such
as Roitt, I. et al., IMMUNOLOGY, C. V. Mosby Co., St. Louis, Mo.
(1985) or Klein, J., IMMUNOLOGY, Blackwell Scientific Publications,
Inc., Cambridge, Mass., (1990).
[0112] In yet another embodiment, for the production of antibodies
that specifically bind one or more outer surface epitopes of OMP21,
intact M. catarrhalis cells or blebs prepared therefrom are used as
immunogen. The cells or blebs may be fixed with agents such as
formaldehyde or glutaraldehyde before immunization. See Harlow and
Lane, Antibodies: A Laboratory Manual, Cold Spring harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1988, Chapter 15. It is
preferred that such anti-whole cell antibodies be monoclonal
antibodies. Hybridoma lines producing the desired monoclonal
antibodies can be identified by using purified OMP21 as the
screening ligand. Cells or blebs of any M. catarrhalis strain
including, but not limited to, ATCC 49143, ATCC 25238, ATCC 25240,
ATCC 43617, ATCC 43618, ATCC 43627 and ATCC 43628 are used as the
immunogen for inducing these antibodies. Preferably, cells or blebs
of strain ATCC 49143 are used as the immunogen for inducing these
antibodies.
[0113] In general, an animal (a wide range of vertebrate species
can be used, the most common being humans, mice, rats, guinea pig,
hamsters and rabbits) is immunized with the OMP21, nucleic acid
sequence thereof or immunogenic fragment or derivative thereof of
the present invention in the absence or presence of an adjuvant or
any agent that would enhance the immunogen's effectiveness and
boosted at regular intervals. The animal serum is assayed for the
presence of desired antibody by any convenient method. The serum or
blood of said animal can be used as the source of polyclonal
antibodies.
[0114] Polyclonal antibodies produced by whole cell or bleb
immunizations contain antibodies that bind other M. catarrhalis
outer membrane proteins ("non-anti-OMP21 antibodies") and thus are
more cumbersome to use where it is known or suspected that the
sample contains other M. catarrhalis outer membrane proteins or
materials that are cross-reactive with these other outer membrane
proteins. Under such circumstances, any binding by the anti-whole
cell antibodies of a given sample or band must be verified by
coincidental binding of the same sample or band by antibodies that
specifically bind OMP21 (e.g., anti-OMP21), or by competition tests
using anti-OMP21 antibodies or OMP21 as the competitor (i.e.,
addition of anti-OMP21 antibodies or OMP21 to the reaction mix
lowers or abolishes sample binding by anti-whole cell antibodies).
Alternatively, such polyclonal antisera, containing
"non-anti-OMP21" antibodies, may be cleared of such antibodies by
standard approaches and methods. For example, the non-anti-OMP21
antibodies may be removed by precipitation with cells of
deletion-mutant M. catarrhalis cultivars or M. catarrhalis strains
known not to have the OMP21; or by absorption to columns comprising
such cells or outer membrane proteins of such cells.
[0115] In further embodiments, useful immunogens for eliciting
antibodies of the invention comprise mixtures of two or more of any
of the above-mentioned individual immunogens, preferred are
mixtures of OMP21 and OMP106.
[0116] Immunization of animals with the immunogens described
herein, preferably humans, rabbits, guinea pigs, chinchillas, rats,
mice, sheep, goats, cows or horses, is performed following
procedures well known to those skilled in the art, for purposes of
obtaining antisera containing polyclonal antibodies or hybridoma
lines secreting monoclonal antibodies.
[0117] Monoclonal antibodies can be prepared by standard
techniques, given the teachings contained herein. Such techniques
are disclosed, for example, in U.S. Pat. No. 4,271,145 and U.S.
Pat. No. 4,196,265. Briefly, an animal is immunized with the
immunogen.
[0118] As a general method for isolating monoclonal antibodies,
when an acceptable antibody titre is detected, the animal is
euthanized and the spleen is aseptically removed for fusion. The
spleen cells are mixed with a specifically selected immortal
myeloma cell line, and the mixture is then exposed to an agent,
typically polyethylene glycol or the like, which promotes the
fusion of cells. Under these circumstances fusion takes place in a
random selection and a fused cell mixture together with unfused
cells of each type is the resulting product. The myeloma cell lines
that are used for fusion are specifically chosen such that, by the
use of selection media, such as HAT: hypoxanthine, aminopterin, and
thymidine, the only cells to persist in culture from the fusion
mixture are those that are hybrids between cells derived from the
immunized donor and the myeloma cells. After fusion, the cells are
diluted and cultured in the selective media. The culture media is
screened for the presence of antibody having desired specificity
towards the chosen antigen. Those cultures containing the antibody
of choice are cloned by limiting dilution until it can be adduced
that the cell culture is single cell in origin. Other methods for
generating monoclonal antibodies are now known and such methods are
included in this invention; for instance, recombinant monoclonal
antibodies selected from recombinant bacteria are also
included.
[0119] Immunization regimens for production of both polyclonal and
monoclonal antibodies are well known in the art. The immunogen may
be administered by any of a number of routes, including
subcutaneous, intravenous, intraperitoneal, intradermal,
intramuscular, mucosal, or a combination of these. The immunogen
may be administered in soluble form, aggregate form, and optionally
attached to or mixed with a physical carrier and/or an adjuvant,
using methods and materials well known in the art. The antisera and
antibodies may be purified using column chromatography methods well
known to those of skill in the art.
[0120] According to the present invention, OMP21s of M. catarrhalis
strains are immuno-cross reactive. Thus, antibodies raised to OMP21
of one M. catarrhalis strain or cultivar specifically bind OMP21 of
other M. catarrhalis strains and cultivars. For example, polyclonal
anti-OMP21 antibodies induced by OMP21 of strain ATCC 49143
specifically bind not only the homologous OMP21 (i.e., the OMP21 of
strain ATCC 49143) but also OMP21 of other M. catarrhalis strains
including, but not limited to, ATCC 43628, ATCC 43627, ATCC 43618,
ATCC 43617, ATCC 25240 and ATCC 25238.
[0121] The antibodies of the invention, including but not limited
to anti-OMP21 antibodies, can be used to facilitate isolation and
purification of OMP21. The antibodies may also be used as probes
for identifying clones in expression libraries that have inserts
encoding OMP21. The antibodies may also be used in immunoassays
(e.g., ELISA, RIA, Westerns) to specifically detect and/or
quantitate M. catarrhalis in biological specimens.
[0122] The antibodies of the invention, particularly those which
are cytotoxic, may also be used in passive immunization to prevent
or attenuate M. catarrhalis infections of animals, including
humans. (As used herein, a cytotoxic antibody is one which enhances
opsonization and/or complement killing of the bacterium bound by
the antibody). An effective concentration of polyclonal or
monoclonal antibodies raised against one or more of the immunogens
of the invention may be administered to a host to achieve such
effects. The exact concentration of the antibodies administered
will vary according to each specific antibody preparation, but may
be determined using standard techniques well known to those of
ordinary skill in the art. Administration of the antibodies may be
accomplished using a variety of techniques, including, but not
limited to those described in Section 5.5. for the delivery of
vaccines.
5.5. Pharmaceutical Compositions
[0123] The present invention also provides pharmaceutical
compositions, including therapeutic and prophylactic compositions,
which may be immunogenic compositions including vaccines, useful
for treating, preventing or ameliorating M. catarrhalis infections
of animals, including humans. Preferred pharmaceutical compositions
are vaccines.
[0124] The pharmaceutical compositions are preferably vaccines, and
can be prepared by techniques known to those skilled in the art and
comprise, for example, an immunologically or therapeutically
effective amount of any of the OMP21 immunogens disclosed in
Section 5.4., optionally in combination with or fused to or
conjugated to one or more other immunogens including lipids,
phospholipids, carbohydrates including lipopolysaccharides, and
other proteins of Moraxella or other bacterial origin, entire
organisms or subunits thereof, a pharmaceutically acceptable
carrier, possibly an appropriate adjuvant, and possibly other
materials traditionally found in pharmaceutical compositions,
including vaccines.
[0125] Preferred other immunogens include any Moraxella, Neisseria,
Pseudomonas, Streptococcus, or Haemophilus attenuated or
inactivated whole organism, or a protein or a carbohydrate
therefrom. More preferred immunogenic compositions, including
vaccines, are cocktail vaccines that comprise OMP21 in combination
with OMP106 and one or more adjuvants, or Hin47 as described in
U.S. Pat. No. 5,679,547 (incorporated herein by reference in its
entirety). Such a cocktail vaccine has the advantage that immunity
against several pathogens can be obtained by a single
administration. Examples of other immunogens are those used in the
known DPT vaccines.
[0126] According to another embodiment, the vaccines of the
invention comprise an immunologically effective amount of any of
the immunogens disclosed in Section 5.4., and additionally an
inactivated or attenuated M. catarrhalis cultivar of the invention.
An inactivated or attenuated M. catarrhalis cultivar is obtained
using any methods known in the art including, but not limited to,
chemical treatment (e.g., formalin), heat treatment and
irradiation. A vaccine which contains antigenic material of only
one pathogen is a monovalent vaccine. Vaccines which contain
antigenic material of several pathogens are combined vaccines and
are also encompassed by the present invention. Such combined
vaccines contain, for example, material from various pathogens or
from various strains of the same pathogen, or from combinations of
various pathogens.
[0127] The vaccine may also contain one or more adjuvants to
improve or enhance or efficiently induce humoral immune responses
(HIR) and cell-mediated immunity (CMI). Adjuvants may act by
retaining the antigen locally near the site of administration to
produce a depot effect facilitating a slow, sustained release of
antigen to cells of the immune system. Adjuvants can also attract
cells of the immune system to an antigen depot and stimulate such
cells to elicit immune responses.
[0128] Desirable characteristics of ideal adjuvants include:
(1) lack of toxicity; (2) ability to stimulate a long-lasting
immune response; (3) simplicity of manufacture and stability in
long-term storage; (4) ability to elicit both CMI and HIR to
antigens administered by various routes, if required; (5) synergy
with other adjuvants; (6) capability of selectively interacting
with populations of antigen presenting cells (APC); (7) ability to
specifically elicit appropriate T.sub.H1 or T.sub.H2 cell-specific
immune responses; and (8) ability to selectively increase
appropriate antibody isotype levels (for example, IgA) against
antigens.
[0129] Immunostimulatory agents or adjuvants have been used for
many years. Intrinsic adjuvants, such as lipo-polysaccharides,
normally are the components of the killed or attenuated bacteria
used as vaccines. Extrinsic adjuvants are immunomodulators which
are typically non-covalently linked to antigens and are formulated
to enhance the host immune responses. Thus, adjuvants have been
identified that enhance the immune response to antigens delivered
parenterally. Aluminum hydroxide and aluminum phosphate
(collectively commonly referred to as alum) are routinely used as
adjuvants in human and veterinary vaccines. The efficacy of alum in
increasing antibody responses to diphtheria and tetanus toxoids is
well established and a HBsAg vaccine has been adjuvanted with alum.
While the usefulness of alum is well established for some
applications, it has limitations. For example, alum is ineffective
for influenza vaccination and inconsistently elicits a cell
mediated immune response.
[0130] Other extrinsic adjuvants may include saponins complexed to
membrane protein antigens (immune stimulating complexes), pluronic
polymers with mineral oil, killed mycobacteria in mineral oil,
Freund's complete adjuvant, bacterial products, such as muramyl
dipeptide (MDP) and lipopolysaccharide (LPS), as well as lipid A,
and liposomes.
[0131] U.S. Pat. No. 4,855,283 granted to Lockhoff et al on Aug. 8,
1989 which is incorporated herein by reference, teaches glycolipid
analogues including N-glycosylamides, N-glycosylureas and
N-glycosylcarbamates, each of which is substituted in the sugar
residue by an amino acid, as immuno-modulators or adjuvants. Thus,
Lockhoff et al. (U.S. Pat. No. 4,855,283), reported that
N-glycosphospholipids and glycoglycerolipids, are capable of
eliciting strong immune responses in both herpes simplex virus
vaccine and pseudorabies virus vaccine. Some glycolipids have been
synthesized from long chain-alkylamines and fatty acids that are
linked directly with the sugars through the anomeric carbon atom,
to mimic the functions of the naturally occurring lipid
residues.
[0132] U.S. Pat. No. 4,258,029 granted to Moloney, (incorporated
herein by reference), teaches that octadecyl tyrosine hydrochloride
(OTH) functioned as an adjuvant when complexed with tetanus toxoid
and formalin inactivated type I, II and III poliomyelitis virus
vaccine. Lipidation of synthetic peptides has also been used to
increase their immunogenicity.
[0133] Therefore, according to the invention, the pharmaceutical
compositions comprising OMP21 may further comprise an adjuvant,
such as, but not limited to alum, QS21, heat labile toxin from
enterotoxigenic E. coli (LT), Cholera toxin (CT), or Bacille
Calmette-Guerine (BCG) and mutated or modified forms of the
above.
[0134] The term "immunologically effective amount" is used herein
to mean an amount sufficient to induce a cellular or humoral immune
response. The amount needed will vary depending upon the
immunogenicity of the OMP21 and the species and weight of the
subject to be vaccinated, but may be ascertained using standard
techniques known in the art in view of the teachings provided
herein. Preferably, the vaccine elicits an immune response in a
subject which produces antibodies including anti-OMP21 antibodies
and more preferably, antibodies that neutralize bacterial binding,
are opsonizing or are bactericidal. More preferably the immune
response is one that can prevent M. catarrhalis infections or
attenuate the severity of any preexisting or subsequent M.
catarrhalis infections. In preferred, non-limiting, embodiments of
the invention, an effective amount of vaccine produces an elevation
of anti-bacterial antibody titer to at least two, more preferably
three, times the antibody titer prior to vaccination.
[0135] In general, the quantity of immunogen will be between 0.1
and 500 micrograms per dose. In a preferred, specific, non-limiting
embodiment of the invention, approximately 0.1 to 100 .mu.g and
preferably 10 to 50 .mu.g are administered to a host. The
compositions of the present invention may also further comprise a
suitable pharmaceutical carrier. The carriers are known to those
skilled in the art and include stabilizers, diluents, excipients
and buffers. Suitable stabilizers include carbohydrates, such as
sorbitol, lactose, mannitol, starch, sucrose, dextran, and glucose
and proteins, such as albumin or casein. Suitable diluents include
saline, Hanks Balanced Salts, and Ringers solution. Such excipients
may include, water, saline, dextrose, glycerol, ethanol, and
combinations thereof. Suitable buffers include an alkali metal
phosphate, an alkali metal carbonate, or an alkaline earth metal
carbonate. Other suitable pharmaceutical carriers are described in
Remington's Pharmaceutical Sciences, Mack Publishing Company, a
standard reference text in this field, which is incorporated herein
by reference in its entirety.
[0136] The vaccines of the invention are prepared by techniques
known to those skilled in the art, given the teachings contained
herein. Generally, an immunogen is mixed with a carrier to form a
solution, suspension, or emulsion. One or more of the additives
discussed above may be in the carrier or may be added subsequently.
The vaccine preparations may be desiccated, for example, by freeze
drying for storage purposes. If so, they may be subsequently
reconstituted into liquid vaccines by the addition of an
appropriate liquid carrier.
[0137] The vaccines are administered to humans or other animals.
They can be administered in one or more doses. Suitable regimes for
initial administration and booster doses are also variable, but may
include an initial administration followed by subsequent
administrations. The dose may also depend on the route of
administration and will vary according to the size of the host.
These compositions can take the form of solutions, suspensions,
tablets, pills, capsules, sustained release formulations or
powders, etc., and may contain about 0.0001 to 95 wt % of the
OMP21, preferably 0.001 to 10 wt %. The vaccines may be
administered by known routes of administration. Many methods may be
used to introduce the vaccine formulations described here. These
methods include but are not limited to intradermal, intramuscular,
intraperitoneal, intravenous, subcutaneous, oral (intragastric),
intranasal, intravaginal or intrarectal routes and other mucosal
routes. Alternatively, other modes of administration including
suppositories and oral formulations may be desirable. The preferred
routes are intramuscular or subcutaneous injection.
5.6. Methods of Detecting
[0138] The OMP21 is useful as an antigen in immunoassays including
enzyme-linked immunosorbent assays (ELISA), RIAs and other
non-enzyme linked antibody binding assays or procedures known in
the art for the detection of anti-bacterial, anti-M. catarrhalis,
and anti-OMP21 protein antibodies in a test sample. In ELISA
assays, the OMP21 is immobilized onto a selected surface, for
example, a surface capable of binding proteins such as the wells of
a polystyrene microtiter plate. After washing to remove
incompletely absorbed OMP21, a nonspecific protein such as a
solution of bovine serum albumin (BSA) that is known to be
antigenically neutral with regard to the test sample may be bound
to the selected surface. This allows for blocking of nonspecific
absorption sites on the immobilizing surface and thus reduces the
background caused by nonspecific binding of antisera onto the
surface.
[0139] The immobilizing surface is then contacted with a test
sample to be tested in a manner conducive to immune complex
(antigen/antibody) formation. Test samples may include human fluids
or solid samples, including but not limited to human blood, serum,
plasma, saliva, urine, stool, sputum, and any other clinically
isolated fluid samples, such as from an infected ear, and/or
biological materials, etc. This may include diluting or
solubilizing the sample with a diluent, such as a solution of BSA,
bovine gamma globulin (BGG) and/or phosphate buffered saline
(PBS)/Tween. The sample is then allowed to incubate for from 2 to 4
hours, at temperatures such as of the order of about 20.degree. to
37.degree. C. Following incubation, the sample-contacted surface is
washed to remove non-immunocomplexed material. The washing
procedure may include washing with a solution, such as PBS/Tween or
a borate buffer. Following formation of specific immunocomplexes
between the test sample and the bound OMP21, and subsequent
washing, the occurrence, and even amount, of immunocomplex
formation may be determined by subjecting the immunocomplex to a
second antibody having specificity for the first antibody. If the
test sample is of human origin, the second antibody is an antibody
having specificity for human immunoglobulins and in general
IgG.
[0140] To provide detecting means, the second antibody may have an
associated activity such as an enzymatic activity that will
generate, for example, a color development upon incubating with an
appropriate chromogenic substrate. Quantification may then be
achieved by measuring the degree of color generation using, for
example, a visible spectrophotometer.
[0141] Another embodiment includes diagnostic kits comprising all
of the essential reagents required to perform a desired immunoassay
according to the present invention. The diagnostic kit may be
presented in a commercially packaged form as a combination of one
or more containers holding the necessary reagents. Such a kit may
comprise a Moraxella bacterium or antigenic portion thereof, a
monoclonal or polyclonal antibody of the present invention in
combination with several conventional kit components. Conventional
kit components will be readily apparent to those skilled in the art
and are disclosed in numerous publications, including Antibodies A
Laboratory Manual (E. Harlow, D. Lane, Cold Spring Harbor
Laboratory Press, 1989) which is incorporated herein by reference
in its entirety. Conventional kit components may include such items
as, for example, microtitre plates, buffers to maintain the pH of
the assay mixture (such as, but not limited to Tris, HEPES, etc.),
conjugated second antibodies, such as peroxidase conjugated
anti-mouse IgG (or any anti-IgG to the animal from which the first
antibody was derived) and the like, and other standard
reagents.
5.7. Nucleic Acids Encoding OMP21
[0142] The present invention also provides nucleic acids encoding
OMP21. The nucleotide sequence comprising the entire OMP21 open
reading frame is depicted in FIG. 3, and SEQ ID NO:6. A deduced
amino acid sequence encoded by the open reading frame of OMP21 is
depicted in FIG. 4, and SEQ ID NO:7.
[0143] Nucleic acids of the present invention can be single or
double stranded. The invention also provides nucleic acids
hybridizable to or complementary to the foregoing sequences. In
specific aspects, nucleic acids are provided which comprise a
sequence complementary to at least 10, 25, 50, 100, 200, or 250
contiguous nucleotides of a nucleic acid encoding OMP21 polypeptide
or an OMP21-derived polypeptide. In a specific embodiment, a
nucleic acid which is hybridizable to a nucleic acid encoding OMP21
polypeptide (e.g., having sequence SEQ. ID. NO.: 10 or 12), or to a
nucleic acid encoding an OMP21-derived polypeptide, under
conditions of low stringency is provided.
[0144] By way of example and not limitation, procedures using such
conditions of low stringency are as follows (see also Shilo and
Weinberg, 1981, Proc. Natl. Acad. Sci. USA 78:6789-6792): Filters
containing DNA are pretreated for 6 h at 40.degree. C. in a
solution containing 35% formamide, 5.times.SSC, 50 mM Tris-HCl (pH
7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 .mu.g/ml
denatured salmon sperm DNA. Hybridizations are carried out in the
same solution with the following modifications: 0.02% PVP, 0.02%
Ficoll, 0.2% BSA, 100 .mu.g/ml salmon sperm DNA, 10% (wt/vol)
dextran sulfate, and 5-20.times.10.sup.6 cpm .sup.32P-labeled probe
is used. Filters are incubated in hybridization mixture for 18-20 h
at 40.degree. C., and then washed for 1.5 h at 55.degree. C. in a
solution containing 2.times.SSC, 25 mM Tris-HCl (pH 7.4), 5 mM
EDTA, and 0.1% SDS. The wash solution is replaced with fresh
solution and incubated an additional 1.5 h at 60.degree. C. Filters
are blotted dry and exposed for autoradiography. If necessary,
filters are washed for a third time at 65-68.degree. C. and
re-exposed to film. Other conditions of low stringency which may be
used are well known in the art (e.g., as employed for cross-species
hybridizations).
[0145] In another specific embodiment, a nucleic acid which is
hybridizable to a nucleic acid encoding OMP21 polypeptide or an
OMP21-derived polypeptide under conditions of high stringency is
provided. By way of example and not limitation, procedures using
such conditions of high stringency are as follows: Prehybridization
of filters containing DNA is carried out for 8 h to overnight at
65.degree. C. in buffer composed of 6.times.SSC, 50 mM Tris-HCl (pH
7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500
.mu.g/ml denatured salmon sperm DNA. Filters are hybridized for 48
h at 65.degree. C. in prehybridization mixture containing 100
.mu.g/ml denatured salmon sperm DNA and 5-20.times.10.sup.6 cpm of
.sup.32P-labeled probe. Washing of filters is done at 37.degree. C.
for 1 h in a solution containing 2.times.SSC, 0.01% PVP, 0.01%
Ficoll, and 0.01% BSA. This is followed by a wash in 0.1.times.SSC
at 50.degree. C. for 45 min before autoradiography. Other
conditions of high stringency which may be used are well known in
the art.
[0146] In another specific embodiment, a nucleic acid which is
hybridizable to a nucleic acid encoding OMP21 polypeptide or an
OMP21-derived polypeptide under conditions of moderate stringency
is provided.
[0147] Various other stringency conditions which promote nucleic
acid hybridization can be used. For example, hybridization in
6.times.SSC at about 45.degree. C., followed by washing in
2.times.SSC at 50.degree. C. may be used. Alternatively, the salt
concentration in the wash step can range from low stringency of
about 5.times.SSC at 50.degree. C., to moderate stringency of about
2.times.SSC at 50.degree. C., to high stringency of about
0.2.times.SSC at 50.degree. C. In addition, the temperature of the
wash step can be increased from low stringency conditions at room
temperature, to moderately stringent conditions at about 42.degree.
C., to high stringency conditions at about 65.degree. C. Other
conditions include, but are not limited to, hybridizing at
68.degree. C. in 0.5M NaHPO.sub.4 (pH7.2)/1 mM EDTA/7% SDS, or
hybridization in 50% formamide/0.25 M NaHPO.sub.4 (pH 7.2)/0.25 M
NaCl/1 mM EDTA/7% SDS; followed by washing in 40 mM NaHPO.sub.4 (pH
7.2)/1 mM EDTA/5% SDS at 42.degree. C. or in 40 mM NaHPO.sub.4
(pH7.2) 1 mM EDTA/1% SDS at 50.degree. C. Both temperature and salt
may be varied, or alternatively, one or the other variable may
remain constant while the other is changed.
[0148] Low, moderate and high stringency conditions are well known
to those of skill in the art, and will vary predictably depending
on the base composition of the particular nucleic acid sequence and
on the specific organism from which the nucleic acid sequence is
derived. For guidance regarding such conditions see, for example,
Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual,
Second Edition, Cold Spring Harbor Press, N.Y., pp. 9.47-9.57; and
Ausubel et al., 1989, Current Protocols in Molecular Biology, Green
Publishing Associates and Wiley Interscience, N.Y.
[0149] Nucleic acids encoding NMAP-derived polypeptides, including
but not limited to fragments or a portion thereof, (see Section
5.2), and OMP21 antisense nucleic acids are additionally provided.
As is readily apparent, as used herein, a "nucleic acid encoding a
fragment or portion of a nucleic acid encoding OMP21 polypeptide or
an OMP21-derived polypeptide" shall be construed as referring to a
nucleic acid encoding only the recited fragment or portion of the
nucleic acid encoding OMP21 polypeptide or an OMP21-derived
polypeptide and not the other contiguous portions of the nucleic
acid encoding OMP21 polypeptide or an OMP21-derived polypeptide
protein as a continuous sequence.
[0150] Also encompassed are nucleotide sequences substantially
homologous to the above described nucleic acids. As used herein a
"substantially homologous" sequence is at least 70%, preferably
greater than 80%, more preferably greater than 90% identical to a
reference sequence of identical size or when the alignment or
comparison is conducted by a computer homology program or search
algorithm known in the art.
[0151] By way of example and not limitation, useful computer
homology programs include the following: Basic Local Alignment
Search Tool (BLAST) (www.ncbi.nlm.nih.gov) (Altschul et al., 1990,
J. Molec. Biol., 215:403-410, "The BLAST Algorithm; Altschul et
al., 1997, Nuc. Acids Res. 25:3389-3402) a heuristic search
algorithm tailored to searching for sequence similarity which
ascribes significance using the statistical methods of Karlin and
Altschul (1990, Proc. Nat'l Acad. Sci. USA, 87:2264-68; 1993, Proc.
Nat'l Acad. Sci. USA 90:5873-77). Five specific BLAST programs are
provided and the BLASTN program compares a nucleotide query
sequence against a nucleotide sequence database. Additional
algorithms which can be useful are the Smith-Waterman and FASTA
algorithms. See Section 5.1.
[0152] In one aspect, the nucleic acids of the invention may be
synthesized using methods known in the art. Specifically, a portion
of or the entire amino acid sequence of OMP21 may be determined
using techniques well known to those of skill in the art, such as
via the Edman degradation technique (see, e.g., Creighton, 1983,
Proteins: Structures and Molecular Principles, W.H. Freeman &
Co., N.Y., pp. 34-49). The amino acid sequence obtained is used as
a guide for the synthesis of DNA encoding OMP21 from
oligonucleotides using conventional chemical approaches or
polymerase chain reaction (PCR) amplification of overlapping DNA
fragments.
[0153] In another aspect, the amino acid sequence may be used as a
guide for the synthesis of degenerate oligonucleotides which in
turn can be used to screen for OMP21 coding sequences in M.
catarrhalis genomic libraries. Such libraries may be prepared by
isolating DNA from cells of any M. catarrhalis strain. Preferably
the DNA used as the source of the OMP21 polypeptide coding
sequence, for both genomic libraries and PCR amplification, is
prepared from cells of any M. catarrhalis strain including, but not
limited to, ATCC 49143, ATCC 25238, ATCC 25240, ATCC 43617, ATCC
43618, ATCC 43627 and ATCC 43628.
[0154] In the preparation of genomic libraries, DNA fragments are
generated, some of which will encode parts or the whole of M.
catarrhalis OMP21. The DNA may be cleaved at specific sites using
various restriction enzymes. Alternatively, one may use DNase in
the presence of manganese to fragment the DNA, or the DNA can be
physically sheared, as for example, by sonication. The DNA
fragments can then be separated according to size by standard
techniques, including but not limited to, agarose and
polyacrylamide gel electrophoresis, column chromatography and
sucrose gradient centrifugation. The DNA fragments can then be
inserted into suitable vectors, including but not limited to
plasmids, cosmids, bacteriophages lambda or T.sub.4, and yeast
artificial chromosome (YAC). (See, for example, Sambrook et al.,
1989, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Glover, D. M.
(ed.), 1985, DNA Cloning: A Practical Approach, MRL Press, Ltd.,
Oxford, U.K. Vol. I, II.) The genomic library may be screened by
nucleic acid hybridization to labeled probe (Benton and Davis,
1977, Science 196:180; Grunstein and Hogness, 1975, Proc. Natl.
Acad. Sci. U.S.A. 72:3961).
[0155] The genomic libraries may be screened with a labeled
degenerate oligonucleotide as a screening probe corresponding to
the amino acid sequence or any complement thereof of any peptide of
OMP21 using optimal approaches well known in the art. or fragments
may be used as the probe. Any probe used preferably is 15
nucleotides or longer.
[0156] Clones in libraries with insert DNA encoding OMP21 will
hybridize to one or more of the degenerate oligonucleotide probes.
Hybridization of such oligonucleotide probes to genomic libraries
are carried out using methods known in the art. For example,
hybridization may be carried out in 2.times.SSC, 1.0% SDS at
50.degree. C. and washed using the same conditions.
[0157] In yet another aspect, clones of nucleotide sequences
encoding a part or the entire OMP21 may also be obtained by
screening M. catarrhalis expression libraries. For example, M.
catarrhalis DNA is isolated and random fragments are prepared and
ligated into an expression vector (e.g., a bacteriophage, plasmid,
phagemid or cosmid) such that the inserted sequence in the vector
is capable of being expressed by the host cell into which the
vector is then introduced. Various screening assays can then be
used to select for the expressed OMP21. In one embodiment, the
various anti-OMP21 antibodies of the invention (see Section 5.5)
can be used to identify the desired clones using methods known in
the art. See, for example, Harlow and Lane, 1988, Antibodies: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., Appendix IV. Clones or plaques from the library are
brought into contact with the antibodies to identify those clones
that bind.
[0158] In an embodiment, colonies or plaques containing DNA that
encodes OMP21 could be detected using DYNA Beads according to
Olsvick et al., 29th ICAAC, Houston, Tex. 1989, incorporated herein
by reference. Anti-OMP21 antibodies are crosslinked to tosylated
DYNA Beads M280, and these antibody-containing beads would then be
used to adsorb to colonies or plaques expressing OMP21 on the
particle or colony surface. Colonies or plaques expressing OMP21
are identified as any of those that bind the beads.
[0159] Alternatively, the anti-OMP21 antibodies can be
nonspecifically immobilized to a suitable support, such as silica
or Celite resin. This material would then be used to adsorb to
bacterial colonies expressing OMP21 as described in the preceding
paragraph.
[0160] In another aspect, PCR amplification may be used to produce
substantially pure DNA encoding a part of or the whole of OMP21
from M. catarrhalis genomic DNA. Oligonucleotide primers,
degenerate or otherwise, corresponding to known OMP21 polypeptide
amino-terminal sequences can be used as 5' primers. Oligonucleotide
sequences, degenerate or otherwise, that are reverse complements of
DNA sequences encoding the carboxy-terminal are used as the 3'
primer.
[0161] PCR can be carried out, e.g., by use of a Perkin-Elmer Cetus
thermal cycler and Taq polymerase (Gene Amp.TM.). One can choose to
synthesize several different degenerate primers, for use in the PCR
reactions. It is also possible to vary the stringency of annealing
conditions used in priming the PCR reactions, to allow for greater
or lesser degrees of nucleotide sequence similarity between the
degenerate primers and the corresponding sequences in M.
catarrhalis DNA. After successful amplification of a segment of the
sequence encoding the OMP21 polypeptide, that segment may be
molecularly cloned and sequenced, and utilized as a probe to
isolate a complete genomic clone. This, in turn, will permit the
determination of the gene's complete nucleotide sequence, the
analysis of its expression, and the production of its protein
product for functional analysis, as described infra.
[0162] Once an OMP21 polypeptide coding sequence has been isolated
from one M. catarrhalis strain or cultivar, it is possible to use
the same approach to isolate OMP21 polypeptide coding sequences
from other M. catarrhalis strains and cultivars. It will be
recognized by those skilled in the art that the DNA or RNA sequence
encoding OMP21 polypeptide (or fragments thereof) of the invention
can be used to obtain other DNA or RNA sequences that hybridize
with it under conditions of moderate to high stringency, using
general techniques known in the art, and as discussed above.
[0163] Hybridization with an OMP21 sequence from one M. catarrhalis
strain or cultivar under high stringency conditions will identify
the corresponding sequence from other strains and cultivars. High
stringency conditions vary with probe length and base composition.
The formula for determining such conditions are well known in the
art. See Sambrook et al., 1989, Molecular Cloning, A Laboratory
Manual, Cold Spring Harbor Press, NY, Chapter 11. As used herein
high stringency hybridization conditions as applied to probes of
greater than 300 bases in length involve a final wash in
0.1.times.SSC/0.1% SDS at 68.degree. C. for at least 1 hour
(Ausubel, et al., Eds., 1989, Current Protocols in Molecular
Biology, Vol. I, Greene Publishing Associates, Inc. and John Wiley
& Sons, Inc., New York, at page 2.10.3). In particular
embodiments, the high stringency wash in hybridization is
2.times.SSC, 1% SDS at 50.degree. C. for about 20 to about 30
minutes.
[0164] One skilled in the art would be able to identify complete
clones of OMP21 polypeptide coding sequence using approaches well
known in the art. The extent of OMP21 polypeptide coding sequence
contained in an isolated clone may be ascertained by sequencing the
cloned insert and comparing the deduced size of the polypeptide
encoded by the open reading frames (ORFs) with that of OMP21 and/or
by comparing the deduced amino acid sequence with that of known
amino acid sequence of purified OMP21. Where a partial clone of
OMP21 polypeptide coding sequence has been isolated, complete
clones may be isolated by using the insert of the partial clone as
hybridization probe. Alternatively, a complete OMP21 coding
sequence can be reconstructed from partial clones by aligning
overlapping inserts.
[0165] Complete clones may be any that have ORFs with deduced amino
acid sequence matching that of OMP21 or, where the complete amino
acid sequence of the latter is not available, that of a peptide
fragment of OMP21 and having a molecular weight corresponding to
that of OMP21. Further, complete clones may be identified by the
ability of their inserts, when placed in an expression vector, to
produce a polypeptide that binds antibodies specific to the
amino-terminal of OMP21 and antibodies specific to the
carboxyl-terminal of OMP21.
[0166] Nucleic acid sequences encoding OMP21 may be produced by
methods well known in the art. In one aspect, sequences encoding
OMP21 can be derived by recombinant DNA methods in view of the
teachings disclosed herein. For example, the coding sequence of
OMP21 may be altered creating amino acid substitutions that will
not affect the immunogenicity of the OMP21 or which may improve its
immunogenicity. Various methods may be used, including but not
limited to oligonucleotide directed, site specific mutagenesis.
These and other techniques known in the art may be used to create,
single or multiple mutations, such as replacements, insertions,
deletions, and transpositions, as described in Botstein and
Shortle, 1985, Science 229:1193-1210.
[0167] Further, DNA of OMP21 coding sequences may be truncated by
restriction enzyme or exonuclease digestions. Heterologous coding
sequence may be added to OMP21 coding sequence by ligation or PCR
amplification. Moreover, DNA encoding the whole or a part of an
OMP21 may be synthesized chemically or using PCR amplification
based on the known or deduced amino acid sequence of OMP21 and any
desired alterations to that sequence.
[0168] The identified and isolated DNA containing OMP21 coding
sequence can be inserted into an appropriate cloning vector. A
large number of vector-host systems known in the art may be used.
Possible vectors include, but are not limited to, plasmids or
modified viruses, but the vector system must be compatible with the
host cell used. Such vectors include, but are not limited to,
bacteriophages such as lambda derivatives, or plasmids such as
pBR322 or pUC plasmid derivatives. The insertion into a cloning
vector can, for example, be accomplished by ligating the DNA
fragment into a cloning vector which has complementary cohesive
termini. However, if the complementary restriction sites used to
fragment the DNA are not present in the cloning vector, the ends of
the DNA molecules may be enzymatically modified. Alternatively, any
site desired may be produced by ligating nucleotide sequences
(linkers) onto the DNA termini; these ligated linkers may comprise
specific chemically synthesized oligonucleotides encoding
restriction endonuclease recognition sequences. In an alternative
method, the cleaved DNA may be modified by homopolymeric tailing.
Recombinant molecules can be introduced into host cells via
transformation, transfection, infection, electroporation, etc., so
that many copies of the gene sequence are generated.
[0169] In an alternative method, the desired DNA containing OMP21
coding sequence may be identified and isolated after insertion into
a suitable cloning vector in a "shot gun" approach. Enrichment for
the desired sequence, for example, by size fractionation, can be
done before insertion into the cloning vector.
[0170] In specific embodiments, transformation of host cells with
recombinant DNA molecules that contain OMP21 coding sequence
enables generation of multiple copies of such coding sequence.
Thus, the coding sequence may be obtained in large quantities by
growing transformants, isolating the recombinant DNA molecules from
the transformants and, when necessary, retrieving the inserted
coding sequence from the isolated recombinant DNA.
5.8. Recombinant Production of OMP21
[0171] OMP21 may be produced through genetic engineering
techniques. In this case, OMP 21 is produced by an appropriate host
cell that has been transformed by DNA that codes for the
polypeptide. The nucleotide sequence encoding OMP21 can be inserted
into an appropriate expression vector, i.e., a vector which
contains the necessary elements for the transcription and
translation of the inserted polypeptide-coding sequence. The
nucleotide sequence encoding OMP21 is inserted into the vectors in
a manner that it will be expressed under appropriate conditions
(e.g., in proper orientation and correct reading frame and with
appropriate expression sequences, including an RNA polymerase
binding sequence and a ribosomal binding sequence).
[0172] A variety of host-vector systems may be utilized to express
the polypeptide-coding sequence. These include but are not limited
to mammalian cell systems infected with virus (e.g., vaccinia
virus, adenovirus, etc.); insect cell systems infected with virus
(e.g., baculovirus); microorganisms such as yeast containing yeast
vectors, or bacteria transformed with bacteriophage DNA, plasmid
DNA, or cosmid DNA. Preferably, the host cell is a bacterium, and
most preferably the bacterium is E. coli, B. subtilis or
Salmonella.
[0173] Plasmid vectors containing replicon and control sequences
which are derived from species compatible with the host cell may be
used for the expression of the genes encoding the OMP21 in
expression systems. Expression vectors contain all the necessary
elements for the transcription and translation of the inserted
protein coding sequence. The vector ordinarily carries a
replication site, as well as marking sequences which are capable of
providing phenotype selection in transformed cells. For example, E.
coli may be transformed using pBR322 which contains genes for
ampicillin and tetracycline resistance cells. Additional examples
include, but are not limited to pTrc99A, pUC19, pUC18, pKK223-3,
pEX1, pCAL, pET, pSPUTK, pTrxFus, pThioHis, pTrcHis, pTrcHis2, and
pLEx. The plasmids or phage, must also contain, or be modified to
contain, promoters which can be used by the host cell for
expression of its own proteins.
[0174] In addition, phage vectors containing replicon and control
sequences that are compatible with the host can be used as a
transforming vector in connection with these hosts. For example,
the phage in lambda GEM.TM.-11 may be utilized in making
recombinant phage vectors which can be used to transform host
cells, such as E. coli LE392.
[0175] Promoters commonly used in recombinant DNA construction
include the .beta.-lactamase (penicillinase) and lactose promoter
systems and other microbial promoters, such as the T7 promoter
system as described in U.S. Pat. No. 4,952,496. Details concerning
the nucleotide sequences of promoters are known, enabling a skilled
worker to ligate them functionally with genes. The particular
promoter used will generally be matter of choice depending upon the
desired results. Hosts that are appropriate for expression of the
OMP21 genes, fragments, analogs or variants thereof, may include E.
coli, Bacillus species, Haemophilus, fungi, yeast, Bordetella, or
the baculovirus expression system may be used.
[0176] The expression elements of vectors vary in their strengths
and specificities. Depending on the host-vector system utilized,
any one of a number of suitable transcription and translation
elements may be used. In a specific embodiment, a chimeric protein
comprising the OMP21 sequence and a pre and/or pro sequence of the
host cell is expressed. In other specific embodiments, a chimeric
protein comprising OMP21 sequence and an affinity purification
peptide is expressed. In further specific embodiments, a chimeric
protein comprising OMP21 and a useful immunogenic peptide or
polypeptide is expressed. In preferred embodiments, expressed OMP21
contains a sequence forming either an outer surface epitope or the
receptor-binding domain of native OMP21.
[0177] Any method known in the art for inserting DNA fragments into
a vector may be used to construct expression vectors containing a
chimeric gene consisting of appropriate
transcriptional/translational control signals and the polypeptide
coding sequences. These methods may include in vitro recombinant
DNA and synthetic techniques and in vivo recombinants (genetic
recombination). Expression of a nucleic acid sequence encoding
OMP21 may be regulated by a second nucleic acid sequence so that
the inserted sequence is expressed in a host transformed with the
recombinant DNA molecule. For example, expression of the inserted
sequence may be controlled by any promoter/enhancer element known
in the art. Promoters which may be used to control expression of
inserted sequences include, but are not limited to the SV40 early
promoter region (Bernoist and Chambon, 1981, Nature 290:304-310),
the promoter contained in the 3' long terminal repeat of Rous
sarcoma virus (Yamamoto et al., 1980, Cell 22:787-797), the herpes
thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad.
Sci. U.S.A. 78:1441-1445), the regulatory sequences of the
metallothionein gene (Brinster et al., 1982, Nature 296:39-42) for
expression in animal cells; the promoters of .beta.-lactamase
(VIIIa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. U.S.A.
75:3727-3731), tac (DeBoer et al., 1983, Proc. Natl. Acad. Sci.
U.S.A. 80:21-25), _P.sub.L, or trc for expression in bacterial
cells (see also "Useful proteins from recombinant bacteria" in
Scientific American, 1980, 242:74-94); the nopaline synthetase
promoter region or the cauliflower mosaic virus 35S RNA promoter
(Gardner et al., 1981, Nucl. Acids Res. 9:2871), and the promoter
of the photosynthetic enzyme ribulose biphosphate carboxylase
(Herrera-Estrella et al., 1984, Nature 310:115-120) for expression
implant cells; promoter elements from yeast or other fungi such as
the Gal4 promoter, the ADC (alcohol dehydrogenase) promoter, PGK
(phosphoglycerol kinase) promoter, alkaline phosphatase
promoter.
[0178] Expression vectors containing OMP21 coding sequences can be
identified by three general approaches: (a) nucleic acid
hybridization, (b) presence or absence of "marker" gene functions,
and (c) expression of inserted sequences. In the first approach,
the presence of a foreign gene inserted in an expression vector can
be detected by nucleic acid hybridization using probes comprising
sequences that are homologous to the inserted OMP21 coding
sequence. In the second approach, the recombinant vector/host
system can be identified and selected based upon the presence or
absence of certain "marker" gene functions (e.g., thymidine kinase
activity, resistance to antibiotics, transformation phenotype,
occlusion body formation in baculovirus, etc.) caused by the
insertion of foreign genes in the vector. For example, if the OMP21
coding sequence is inserted within the marker gene sequence of the
vector, recombinants containing the insert can be identified by the
absence of the marker gene function. In the third approach,
recombinant expression vectors can be identified by assaying the
foreign gene product expressed by the recombinant. Such assays can
be based, for example, on the physical or functional properties of
OMP21 in in vitro assay systems, e.g., binding to an OMP21 ligand
or receptor, or binding with anti-OMP21 antibodies of the
invention.
[0179] Once a particular recombinant DNA molecule is identified and
isolated, several methods known in the art may be used to propagate
it. Once a suitable host system and growth conditions are
established, recombinant expression vectors can be propagated and
prepared in quantity. As explained above, the expression vectors
which can be used include, but are not limited to, the following
vectors or their derivatives: human or animal viruses such as
vaccinia virus or adenovirus; insect viruses such as baculovirus;
yeast vectors; bacteriophage vectors (e.g., lambda), and plasmid
and cosmid DNA vectors, to name but a few.
[0180] In addition, a host cell strain may be chosen which
modulates the expression of the inserted sequences, or modifies and
processes the gene product in the specific fashion desired.
Expression from certain promoters can be elevated in the presence
of certain inducers; thus, expression of the genetically engineered
OMP21 may be controlled. Furthermore, different host cells have
characteristic and specific mechanisms for the translational and
post-translational processing and modification of proteins.
Appropriate cell lines or host systems can be chosen to ensure the
desired modification and processing of the foreign protein
expressed.
5.9. Applications
[0181] The present invention has many utilities. By way of example
and not as limiting the invention, the OMP21, antibodies and
nucleic acids of the invention are useful as reagents for clinical
or medical diagnosis of M. catarrhalis infections and for
scientific research on the properties of pathogenicity, virulence,
and infectivity of M. catarrhalis, as well as host defense
mechanisms. For example, DNA and RNA of the invention can be used
as probes to identify the presence of M. catarrhalis in biological
specimens by hybridization or PCR amplification. The DNA and RNA
can also be used to identify other bacteria that might encode a
polypeptide related to the M. catarrhalis OMP21.
[0182] OMP21 of the invention may be used to prepare polyclonal and
monoclonal antibodies that can be used to further purify
compositions containing the polypeptides of the invention by
affinity chromatography. The polypeptides and peptides can also be
used in standard immunoassays to screen for the presence of
antibodies to M. catarrhalis in a sample. The cytotoxic antibodies
of the invention are useful in passive immunizations against M.
catarrhalis infections. OMP21 and nucleic acids encoding same may
further be used as active ingredients in pharmaceutical
compositions, including vaccines, to treat or prevent M.
catarrhalis infections.
[0183] It is to be understood that the application of the teachings
of the present invention to a specific problem or environment will
be within the capabilities of one having ordinary skill in the art
in light of the teachings contained herein.
[0184] The following examples are presented solely for the purpose
of illustration and are not intended to limit the scope of the
invention. Changes in form and substitution of equivalents are
contemplated as circumstances suggest or render expedient. Although
specific terms have been employed herein, such terms are intended
in a descriptive sense and not for purposes of limitation.
[0185] Methods of molecular genetics, protein biochemistry and
immunology used but not explicitly described in the disclosure and
examples are amply reported in the scientific literature and are
well within the ability of those skilled in the art.
6. EXAMPLE
Isolation and Characterization of the OMP21 Polypeptide and Gene
Encoding Same
6.1. Materials and Methods
[0186] 6.1.1. Detergent Extraction of OMP21
[0187] Strains of M. catarrhalis were each grown at 35.degree. C.
at 200 rpm in 1 liter of Mueller Hinton broth in a 4 liter flask.
Outer membrane protein (OMP) preparations were isolated by treating
50 mg of cells (wet weight) with 0.67 ml of 1.5% n-octyl
.beta.-D-glucopyranoside (i.e., octyl glucoside; Oq) or
EmpigenB.TM. (N-dodecyl-N,N-dimethyl-glycine, CalBiochem) in
phosphate buffered saline (PBS; for 30 minutes at room temperature.
Cells were pelleted in a microcentrifuge for 5 minutes and the
supernatant was used as the detergent extract. Comparison of
protein profiles of these extracts from a number of strains of M.
catarrhalis to those of blebs (i.e., outer membrane vesicles)
isolated by differential centrifugation, which are highly enriched
for outer membrane proteins (OMPs) from M. catarrhalis (Murphy and
Loeb, 1989, Microbial Pathogen. 6:159-174) indicates the detergent
extracts contain predominately outer membrane proteins of M.
catarrhalis (FIG. 1). This indicated that detergent extraction
provided a more rapid procedure with a higher yield of outer
membrane proteins as compared to outer membrane proteins prepared
from blebs.
[0188] 6.1.2. Amino Terminal Sequencing of OMP21
[0189] M. catarrhalis ATCC 49143 were grown in Mueller Hinton broth
at 37.degree. C., cells were harvested and suspended in PBS
containing 1 mM magnesium sulfate. The suspension was sonicated and
centrifuged at low speed. The supernatant was centrifuged at high
speed and the pellet collected. The pellet was washed twice using a
high speed centrifugation. The resuspended pellet was mixed with
PAGE sample buffer containing SDS, and was incubated for 5 minutes
in boiling water bath. The proteins were then resolved on a 12% PA
with SDS and transferred to a PVDF membrane by electroblotting,
then stained with coomassie blue R-250. The region of the membrane
containing the OMP21 band was then cut out for amino-terminal
sequencing.
[0190] 6.1.3. Anti-OMP21 Antiserum
[0191] Antiserum to OMP21 were prepared by resolving OMP21
polypeptide from OG extracts of M. catarrhalis strain ATCC 49143 in
a DEAE SEPHAROSE.TM. ion exchange chromatography column. The
fraction containing OMP21 was injected into a rabbit to generate
antiserum to OMP21 polypeptide. In addition, affinity purified
antibody was prepared by injecting rabbits with blebs from
Moraxella catarrhalis and purified using a cyanogen bromide
activated agarose gel with immobilized OMP21. The gel was reacted
with the antiserum and non-reactive antibodies and proteins were
washed from the gel. Reactive antibodies were eluted from the gel
using 100 mM glycine, pH 2.5. The eluted antibodies were washed
with PBS and concentrated. The concentrate was further purified by
reacting with OMP21-deletion mutants of M. catarrhalis. The
antiserum was analyzed by Western blots as described in Section
6.1.4., examined for complement-mediated cytotoxic activity against
M. catarrhalis as described in Section 7 and inhibition of
nasopharyngeal binding as described in Section (infra).
[0192] 6.1.4. Western Blots
[0193] M. catarrhalis were grown in Meuller Hinton broth for 48
hours at 35.degree. C. in 5% CO.sub.2. Cells were collected by
centrifugation and outer membrane proteins were extracted with OG.
Extracts were then mixed by suspending in 150 .mu.l of PAGE sample
buffer (360 mM Tris buffer [pH 8.8], containing 4% sodium
dodecylsulfate and 20% glycerol), and incubating the suspension at
100.degree. C. for 5 minutes. The solubilized cells were resolved
on 4-20% gradient polyacrylamide gels as per Laemmli and the
separated proteins were electrophoretically transferred to PVDF
membranes at 100 V for 1.5 hours as previously described (Thebaine
et al. 1979, Proc. Natl. Acad. Sci. USA 76:4350-4354) except 0.05%
sodium dodecylsulfate was added to the transfer buffer to
facilitate the movement of proteins from the gel. The PVDF
membranes were then pretreated with 25 ml of Dulbecco's phosphate
buffered saline containing 0.5% sodium casein, 0.5% bovine serum
albumin and 1% goat serum. All subsequent incubations were carried
out using this pretreatment buffer.
[0194] PVDF membranes were incubated with 25 ml of a 1:500 dilution
of serum from a rabbit immunized with OMP21 polypeptide (as
described above) for 1 hour at room temperature. PVDF membranes
were then washed twice with wash buffer (20 mM Tris buffer [pH
7.5.] containing 150 mM sodium chloride and 0.05% Tween-20). PVDF
membranes were incubated with 25 ml of a 1:5000 dilution of
peroxidase-labeled goat anti-rabbit IgG (Jackson ImmunoResearch
Laboratories, West Grove Penn. Catalog number 111-035-003) for 30
minutes at room temperature. PVDF membranes were then washed 4
times with wash buffer, and were developed with 3,3'
diaminobenzidine tetrahydrochloride and urea peroxide as supplied
by Sigma Chemical Co. (St. Louis, Mo. catalog number D-4418) for 4
minutes each.
[0195] 6.1.5. Outer Surface Localization of OMP21
[0196] M. catarrhalis ATCC 49143 was grown overnight at
35-37.degree. C. in a shaking water bath in Mueller Hinton broth.
The cells were pelleted by centrifugation and then resuspended in
an equal volume of Dulbecco's modification of phosphate buffered
saline without calcium or magnesium. Cells were diluted in a
carbonate coupling buffer (50 mM sodium bicarbonate, pH 9.6),
aliquots were added to wells in a 96-well ELISA plate and stored
overnight at 2-8.degree. C. The next day, the plates were washed
with PBS/Tween, incubated with a non-specific protein blocker, then
washed again. The wells were then treated with 100 .mu.l of various
dilutions of anti-OMP21 antiserum, or preimmune serum from the same
animal, diluted in PBS/Tween, or PBS for 2 hours, then washed 3
times with PBS/Tween. The wells were treated with 100 .mu.l of
diluted peroxidase-labeled goat antibody to mouse or rabbit IgG
(Jackson ImmunoResearch Laboratories, catalog #111-035-003). The
wells were incubated for 1 hour and washed 3 times in PBS/Tween.
Peroxidase substrate (Kirkegaard and Perry Laboratories, Inc,
Gaithersburg, Md. catalog number 50-76-00) was added to each well
and the reaction incubated for 10 minutes. Substrate stop solution
(Kirkegaard and Perry Laboratories, Inc, Gaithersburg, Md. catalog
number 50-85-05) was added and the absorbance at 450 nm was
determined for each well.
6.2. Results
[0197] 6.2.1. Outer Surface Location of OMP21
[0198] Mouse anti-OMP21 antiserum was used in ELISA to determine if
OMP21 polypeptide is exposed on the outer surface of M. catarrhalis
cells. Whole M. catarrhalis cells reacted with anti-OMP21 antiserum
whereas cells treated with preimmune serum or PBS did not. This
indicates that in intact M. catarrhalis cells OMP21 polypeptide is
reactive with anti-OMP21 antibodies. This result indicates that
OMP21 polypeptide is exposed on the outer surface of M.
catarrhalis. This finding is consistent with OMP21 polypeptide
having a role in adherence or nasopharyngeal binding, and moreover,
indicates that OMP21 polypeptide is useful as a vaccine.
[0199] 6.2.2. Properties of OMP21 Polypeptide
[0200] OMP21 polypeptide is an outer membrane protein. This
conclusion is supported by the finding that extracting M.
catarrhalis cells with detergent solubilizes OMP21 polypeptide.
[0201] Using octyl glucoside extracts of M. catarrhalis, then
incubating the extracts with sodium dodecyl sulfate at 100.degree.
C., and resolving the proteins on a denaturing polyacrylamide gel,
we have estimated the apparent molecular weight of OMP21
polypeptide from various strains of M. catarrhalis, specifically
those of ATCC 25238, ATCC 25240, ATCC 43617, ATCC 43618, ATCC 43627
and ATCC 43628, to range from about 16 kD to about 20 kD (FIG.
2).
[0202] OMP21 polypeptide of strain ATCC 49143 was extracted from
the gel slice and was sequenced. N-terminal sequencing of the
mature OMP21 polypeptide isolated from the outer membrane of ATCC
49143 yielded the following sequence:
TABLE-US-00002 (SEQ ID NO: 1)
AISYGNSADAQPYVGAKIGQVDAKQINGKNTAYGIYAGYN.
[0203] 6.2.3. Conservation of OMP21 Polypeptide
[0204] Western blot analysis of outer membrane protein extracts of
a number of M. catarrhalis strains and related species of bacteria
showed that the anti-OMP21 antibodies bind to a polypeptide of
about 16 kD to about 20 kD in many M. catarrhalis strains (FIG. 8).
These results demonstrate the following: 1) Anti-OMP21 antibodies
may be used to specifically identify and distinguish M. catarrhalis
from related species of bacteria. See FIG. 8 in which antibodies
are useful to distinguish M. catarrhalis from mutants that have a
"knock-out" of OMP21. 2) OMP21 polypeptide may be used to generate
antibodies that have diagnostic application for identification of
M. catarrhalis. 3) Antibodies to OMP21 polypeptide of one strain
(e.g., OMP21 of ATCC 49143) may be used to identify and isolate the
corresponding OMP21 polypeptide of other M. catarrhalis
strains.
7. EXAMPLE
Efficacy of OMP21 Vaccine: Cytotoxic Activity of Anti-OMP21
Antiserum
[0205] Complement-mediated cytotoxic activity of anti-OMP21
antibodies was examined to determine the vaccine potential of OMP21
polypeptide. Affinity purified antibodies to OMP21 from ATCC 49143
were prepared as described in Section 6.1.4. supra. The activities
of the pre-immune serum and the anti-OMP21 antiserum in mediating
complement killing of M. catarrhalis were examined using the "Serum
Bactericidal Test" described by Zollinger et al. (Immune Responses
to Neisseria meningitis, in Manual of Clinical Laboratory
Immunology, 3rd ed., pg 347-349), except that cells of M.
catarrhalis strains were used instead of Neisseria meningitis
cells.
[0206] The results show that anti-OMP21 antiserum mediated
complement-killing of M. catarrhalis ATCC 49143 but not of a
deletion mutant of M. catarrhalis with the OMP21 gene
disrupted.
8. EXAMPLE
Isolation of the omp21 Gene
8.1. Preparation of Primers
[0207] Degenerate PCR primers were designed based on the OMP21
N-terminal sequence information, including the 40 amino acid
sequence depicted in SEQ ID NO:1. The sequence of these degenerate
oligonucleotide primers is as follows:
TABLE-US-00003 SEQ ID NO: 2 GAY GCN CAR CCN TAY GT (128 fold
degeneracy) SEQ ID NO: 3 TGY TTN GCR TCN ACY TG (128 fold
degeneracy) SEQ ID NO: 4 GCN GAY GCN CAR CCN TAY GT (512 fold
degeneracy) SEQ ID NO: 5 ATN CCR TAN GCN GTR TTY TT (512 fold
degeneracy)
[0208] PCR reactions (50 ul) contained 1 ug of M. catarrhalis
genomic DNA, prepared by methods well known to those skilled in the
art, the respective oligonucleotide primers at a final
concentration of 0.5 uM, dNTPs at 0.2 mM, usually either 2 or 4 mM
Mg++, and 2 units of Taq Polymerase. PCR was performed in an Idaho
Rapidcyler using the following cycling program:
Hold 1: 94.degree. C., 1 min; Cycles 1-3: Denature 94.degree. C.,
Anneal 55.degree. C., Elongate 72.degree. C. each for 30 sec for 3
cycles; Cycle 4: Denature 94.degree. C., Anneal 40.degree. C.,
Elongate 72.degree. C. each for 30 sec for 35 cycles;
Hold 2: 72.degree. C., 1 min.
[0209] When used to program a PCR reaction with M. catarrhalis
genomic DNA as a template, these primer pairs generated DNA
fragments of 50 and 80 bp, respectively, as predicted. These
fragments were amplified from the same gene locus as determined by
using the 80 bp DNA fragment as the template in a PCR reaction with
the primer pair of SEQ ID Nos: 2 and 3 to amplify the 50 bp DNA
fragment described above. The 80 bp DNA fragment has the sequence
depicted in SEQ ID NO:21.
[0210] PCR reactions with non-degenerate primers were performed
using the same template, primer and Mg++ conditions. The specific
annealing temperature of an oligonucleotide primer pair was
calculated and used throughout 35 cycles of amplification. The
extension times were adjusted according to the length of the
amplified DNA fragment.
8.2. Suppression PCR
[0211] Suppression PCR was performed using the reagents of the
Universal Genome Walker Kit and the Tth Polymerase Mix (Clontech).
Nested gene-specific primers were designed based on the consensus
sequence for the 80 bp amplification product from the degenerate
primer PCR. Nested oligonucleotide primers (28 mer) designed for
this walk were as follows:
TABLE-US-00004 (SEQ ID NO: 8) CCC TAT GTT GGT GCC AAA ATT GGT CAA G
(SEQ ID NO: 9) AGA TGC CAA GCA AAT CAA CGG TAA GAA C (SEQ ID NO:
10) GTT CTT ACC GTT GAT TTG CTT GGC ATC T (SEQ ID NO: 11) CTT GAC
CAA TTT TGG CAC CAA CAT AGG G
[0212] Initial amplifications were done with combinations of the
anchor primer API from the kit and SEQ ID NOs: 8 and 10,
respectively. The PCR reactions (50 ul) contained 5 ng of DNA
digested to completion with a number of six base pair blunt end
cutters and subsequently ligated to the genome walker adaptor. Mg++
was 1.1 mM and dNTPs were used at a concentration of 0.2 mM. The
cycling conditions for the primary PCR were as follows:
Cycle 1: Denature 94.degree. C., 2 sec, Anneal and Elongate
72.degree. C., 3 min for 7 cycles; Cycle 2: Denature 94.degree. C.,
2 sec. Anneal and Elongate 67.degree. C., 3 min for 32 cycles;
Hold: 67.degree. C., 4 min.
[0213] For the secondary PCR reaction, 1 ul of a 1/50 dilution of
the primary PCR reaction was used as the template. Reactants and
cycling parameters were as above with the exception that the nested
anchor primer 2 from the kit was used in combination with the
nested gene specific primers SEQ ID NOs: 9 and 11,
respectively.
8.3. Isolation and Subcloning of the PCR Product
[0214] The PCR products generated with degenerate oligonucleotide
primers were separated on a 3.5% NuSieve agarose gel (FMC
Bioproducts) using a 20 bp DNA ladder (Invitrogen) as a size
marker. Relevant DNA bands were excised from the gel and recovered
on Geneclean Glassmilk (BIO 101). The same procedure was used to
recover the products of the suppression PCR from a 1% agarose gel.
All gel purified PCR products were ligated with EcoRV digested
pBluescript II SK (20 ng) and electroporated into TOP F' E. coli
competent cells. After recovery on SOC medium (BRL) for 1 hr @
37.degree. C., aliquots of the culture were plated on
LB/x-gal/IPTG/Amp plates and grown over night at 37.degree. C.
8.4. Identification of Recombinant Plasmids
[0215] White colonies from the transformation were picked directly
into a PCR reaction that was primed with commercially available T7
and T3 promoter oligonucleotides. Insertless colonies gave rise to
a .sup..about.160 bp DNA amplification product, whereas plasmids
with the 50 bp and 80 bp PCR amplification products yielded band of
210 and 240 bp in this assay, respectively. Several positive
colonies were grown and high quality plasmid DNA was prepared by
methods known to those skilled in the art (described in laboratory
handbooks such as Molecular Cloning).
8.5. Sequence Analysis
[0216] The sequence of the inserts in recombinant plasmids was
determined using the fluorescent dideoxy-termination method.
Reactions were analyzed on a ABI Prism 310 Genetic Analyzer. The 50
and 80 bp amplification products of the degenerate oligonucleotide
primers were sequenced from both strands using the T3 and T7
promoter primers. The 500 and 1000 bp DNA fragments from the
suppression PCR were initially sequenced with the same primers. In
order to obtain the sequence of the complete omp21 ORF, several
gene specific primers were synthesized and used to corroborate the
sequence. The sequence of these oligonucleotides is shown
below:
TABLE-US-00005 GCG ACA AAA CCA GCC TAG (SEQ ID NO: 12) GGT GTT GGT
GTT GGC TTT (SEQ ID NO: 13) CCC CTT TAA AAC ATC GCC AC. (SEQ ID NO:
14)
[0217] The nucleotide sequence of the entire omp21 gene is shown in
FIG. 3 and is identified as SEQ ID NO:6. A deduced amino acid
sequence of the open reading frame of OMP21 is shown FIG. 4, and is
identified as SEQ ID NO:7.
9. EXAMPLE
Preparation of Recombinant OMP21
9.1. Construction of an Expression Vector
[0218] To facilitate the cloning into the expression plasmid pTrc
99A (Pharmacia), a NcoI site was introduced into the OMP21 ORF at
the start methionine. This change in the DNA sequence affects the
second codon of the OMP 21 ORF as well by changing lysine to
glutamic acid. To make this change more conservative the second
codon was mutated to encode alanine. Mutations at the 3' end of the
OMP21 extended the ORF by a stretch of six histidines, followed by
a translational stop and a HindIII restriction site. These changes
were introduced entirely by PCR using the composite primers shown
below and M. catarrhalis genomic DNA as the template.
TABLE-US-00006 (SEQ ID NO: 15) gga cgc cat ggc aAC TTT AAA AAC ACT
ATT GGC AGT ATC AGC TTC (SEQ ID NO: 16) atc aag ctt agt gat ggt gat
ggt gat gAA AAG CCA AAT GAG CGC
[0219] The resulting expression construct is designated as pOMP21x.
The mutations described above were verified by sequencing the
modified 5' and 3' ends of the insert.
9.2. Expression of OMP21
[0220] E. coli containing the expression plasmid pOMP21x was grown
in L-broth containing 100 mg/ml of ampicillin at 34.degree. C. to
an absorbance at 550 nm of 0.6, then isopropylthio
.beta.-galactosidase was added to a concentration of 1 mM. The
culture was allowed to continue to grow for 3 h. The cells were
collected by centrifugation at 5000 g for 10 min. The cells (1 g)
were suspended to 10% (w/v) on PBS. OMP21 from these cells was
prepared by detergent extract (as described in Section 6.1.2.) and
SDS-PAGE as described in Section 6.1.4.
10. EXAMPLE
Verification of the omp21 Gene
10.1. Construction of an omp21 Gene-Targeting Cassette
[0221] A gene targeting cassette was assembled from two PCR
amplified regions of the omp 21 gene and a Kanamycin Resistance
GenBlock.TM. (Pharmacia). The targeting region 5' to the Kanamycin
gene was amplified using the primer pair SEQ ID NO 17 and SEQ ID NO
18. These composite primers amplify a 20-550 bp DNA fragment from
genomic M. catarrhalis DNA and introduce a SmaI and a PstI
restriction site, respectively, at the end of the fragment. The
targeting region 3' from the Kanamycin gene was amplified in the
same manner using the primers SEQ ID NO 19 and SEQ ID NO 20. These
composite primers amplify a .sup..about.1 kb DNA fragment that has
PstI and SalI sites, respectively, at the ends. The conditions for
the PCR amplification were as follows:
Hold 1: 94.degree. C. for 30 sec; Cycle: Denature 94.degree. C. for
10 sec, Anneal 60.degree. C. for 15 sec, Elongate 72.degree. C. for
45 sec, for 35 cycles;
Hold 2: 72.degree. C. for 1 min.
[0222] Sequence of the PCR primers (restriction sites introduced
are underlined):
TABLE-US-00007 (SEQ ID NO: 17)
gacggcccgggCTGGTATCAATTGGCATAGGCGGTAAGTT (SEQ ID NO: 18)
catgctgcagCTTGACCAATTTTGGCACCAACATAGGG (SEQ ID NO: 19)
cactctgcagTAGACGCCAAGCAAATCAACGGTAAGAACA (SEQ ID NO: 20)
gcatgtcgacGTAGATGAGCTACAAGGCGTGATTTGGGAT.
[0223] The amplified DNA fragments were digested with SmaI and PstI
or PstI and SalI, respectively. The 0.5 kb SmaI/PstI DNA fragment
was cloned into the plasmid pCR-Script AMP SK(+) (Stratagene) in
the same cloning sites. Miniprep DNA from a recombinant carrying
this insert was then restricted with PstI and SalI and ligated to
the 1 kb PstI/SalI insert. A recombinant carrying both flanking
regions was then linearized with Pst I and the Kanamycin cassette
was inserted as a .sup..about.1.2 kb PstI insert. To test for
functionality the transformed bacteria were plated on Kan (50
ug/ml) LB agar. Plasmids isolated from the resulting
Kanamycin-resistant colonies were analyzed for the orientation of
the Kanamycin insert by restriction digestion with Cla I and Sal I.
Cla I cuts once in the 5' flanking 0.5 kb fragment and
asymmetrically in the Kanamycin resistance gene. The resulting
plasmid targeting constructs were thus designated as pomp21 K.O.
(the Kanamycin resistance gene and omp21 are transcribed in the
same direction) or as pomp21 O.K. (Kanamycin and omp 21
transcription proceed towards each other).
10.2 Preparation of Competent Moraxella Catarrhalis Cells
[0224] Moraxella catarrhalis cells were grown to an optical density
(OD600 nm) of 1, harvested by centrifugation (3000.times.g), and
subsequently washed twice in ice-cold distilled water and once in
15% glycerol. The final cell pellet was resuspended in 1-2 ml of
15% glycerol and rapidly frozen in 100 .mu.l aliquots on dry ice.
The electrocompetent cells were stored at -80.degree. C.
10.3. Electroporation of Competent Cells
[0225] Aliquots (50 .mu.l) of electrocompetent cells were mixed
with 1 .mu.g of plasmid DNA, transferred to a 0.1 cm
electroporation cuvette and kept on ice for 1 min. An
electroporation pulse was subsequently delivered using the
following settings: 1500 V., 50.degree. F. and 150.mu.. The pulsed
culture was immediately transferred to Mueller-Hinton medium and
incubated for 6 hrs at 37.degree. C. Aliquots of the culture were
then spread on selective media plates (Mueller-Hinton with 5
.mu.g/ml of Kanamycin) and incubated at 37.degree. C. until
colonies were clearly visible (24-36 hrs). A random sample of
bacteria was picked and restreaked to obtain single colonies.
Individual colonies were grown in 2 ml cultures as above and used
to prepare genomic DNA for PCR analysis.
10.4. PCR Analysis of Putative OMP21 Deletion Mutants
[0226] DNA from KAN.sup.R Moraxella catarrhalis colonies was
analyzed by PCR using the primer pair SEQ ID NO: 17 and SEQ ID NO:
18. The PCR conditions were as above with the exception that the
elongation time was 2 min as was the final extension (Hold 2).
These primers amplify a .sup..about.1.6 kb DNA fragment from wt DNA
and a .sup..about.2.9 kb DNA fragment from the knock-out construct.
These primers were only used for the initial screening of putative
knock-outs. By nature and location of the primers, this PCR
analysis could not distinguish between random or homologous
integrants. Clones that scored positive in this screen were further
analyzed by Southern blot.
10.5. Southern Analysis of OMP 21 Deletion Mutants
[0227] M. catarrhalis DNA was digested with Pst I. The digests were
separated on a 0.8% TAE-agarose gel and transferred to nylon
membranes using standard protocols. The blots were hybridized with
a .sup.32P labeled probe prepared from a 543 bp NotI/ClaI insert
released from the plasmid pCRScript-omp21. Using this probe, a
.sup..about.8 kb DNA fragment is detected in PstI digests of
wild-type M. catarrhalis DNA. The same probe detects a
.sup..about.4.5 kb restriction fragment in all the knock-outs. The
results are shown in FIG. 6. This then constitutes proof that the
gene locus has been altered by homologous recombination.
11. EXAMPLE
RFLP Analysis of omp 21
[0228] Genomic DNA from wild-type Moraxella catarrhalis was
digested with HindIII. The digests were separated on a 0.8%
TAE-agarose gel and transferred to nylon membranes using standard
protocols. The blots were hybridized with a .sup.32P labeled probe
prepared from the ClaI to NotI fragment of the cloning vector which
contained a sequence spanning the omp21 coding region. The high
stringency wash was 2.times.SSC, 1% SDS at 50.degree. C. for about
20 to about 30 minutes. A single 1.8 kb fragment was detected from
all strains tested. The results are shown in FIG. 5. The uniformity
of the RFLP pattern shows that OMP21 gene is highly conserved in
Moraxella catarrhalis.
12. EXAMPLE
Generation and Reactivity OF Monoclonal Anti-OMP21 Antibodies
[0229] BALB/c mice are immunized with total outer membranes from M.
catarrhalis. Hybridomas for monoclonal antibodies are prepared by
fusing the spleen cells from these mice to SP2/0 cells and
selecting for successful hybrids with HAT containing media.
Reactive hybridomas are screened using an ELISA containing
detergent extracts of the total outer member of M. catarrhalis
MC2926. From this screen, hybridomas with varying levels of
activity in the ELISA are selected for clonal selection, the
monoclonal antibodies are assayed for reactivity to purified OMP21
and total outer membranes from M. catarrhalis MC2926_OMP21 by
ELISA.
13. EXAMPLE
Nasopharyngeal Cell Binding
[0230] The binding of Moraxella to the continuous cell line HEp-2
was assayed using a modification of the procedure described by
Galan and Curtiss (J. E. Galan and R. Curtiss III. 1989, Proc.
Natl. Acad. Sci. USA 86:6383-6387, incorporated herein by reference
in its entirety). The M. catarrhalis strains MC2926 and MC2954 were
used to assay the binding of Moraxella to HEp-2 cells. The MC2954
strain is an isogeneic strain to MC2926 but with the gene for OMP21
disrupted (as described in Example in Section 8 above), thereby
causing the loss of the expression of the OMP21 protein.
[0231] Briefly, the strains were grown to mid-log phase in Mueller
Hinton broth. Bacterial cells from the culture were then
centrifuged onto the monolayer of HEp-2 cells and allowed to bind
to the cells for 1 hour. Nonbound cells were removed by washing
with Hanks balanced salt solution containing calcium. Adherent
cells were removed with the monolayer by treatment with 0.1% sodium
glycocholate in phosphate buffered saline (PBS). The number of
adherent cells were enumerated by plating on Mueller Hinton agar
and allowing the bacteria to grow for 24 hours. The efficiency of
binding of the bacteria is expressed as a percentage of bacteria
bound relative to the original number of bacteria added to the
HEp-2 monolayer, and is shown in Table 1 below.
TABLE-US-00008 TABLE 1 Binding Efficiency Of MC2926 And The Genetic
Deletion Of omp 21 (MC2954) To HEp-2 Cells Bacterial strain % bound
MC2926 100% MC2954 45%
The results of the nasopharyngeal cell binding assay show that
OMP21 is responsible for binding and adherence of Moraxella
catarrhalis to nasopharyngeal cells.
14. DEPOSIT OF MICROORGANISM
[0232] E. coli Top10F' containing plasmid OMP21X (pOMP21X), that
contains the open reading frame of the gene encoding the OMP21 of
M. catarrhalis as are described and referred to herein was
deposited on Sep. 16, 1998, with the American Type Culture
Collection (ATCC) located at 10801 University Boulevard, Manassas
Va., 20110-2209, USA, pursuant to the Budapest Treaty and pursuant
to 37 CFR 1.808 and prior to the filing of this application and
assigned accession No. ______. Samples of the deposited materials
will become available to the public upon grant of a patent based
upon this United Stated patent application.
[0233] The present invention described and claimed herein is not to
be limited by the scope of the microorganisms or plasmids
deposited, since the deposited embodiment is intended only as an
illustration of the invention. Any functionally equivalent or
similar microorganisms or plasmids that encode similar or
equivalent proteins or fragments or analogs thereof as described in
this application are intended to be within the scope of the
invention. Indeed, various modifications of the invention, in
addition to those shown and described herein, will become apparent
to those skilled in the art from the foregoing description and
accompanying drawings. Such modifications are intended to fall
within the scope of the appended claims.
[0234] Other equivalents of the present invention may be readily
determined by those skilled in the art and such equivalents are
intended to be included in this invention. The foregoing disclosure
includes all the information deemed essential to enable those
skilled in the art to practice the claimed invention without undue
experimentation. Because the cited patents or publications may
provide further useful information, the disclosures of any and all
cited materials are hereby incorporated by reference herein in
their ehtireties.
Sequence CWU 1
1
21140PRTMoraxella catarrhalis 1Ala Ile Ser Tyr Gly Asn Ser Ala Asp
Ala Gln Pro Tyr Val Gly Ala1 5 10 15Lys Ile Gly Gln Val Asp Ala Lys
Gln Ile Asn Gly Lys Asn Thr Ala20 25 30Tyr Gly Ile Tyr Ala Gly Tyr
Asn35 40217DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 2gaygcncarc cntaygt 17317DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
3tgyttngcrt cnacytg 17420DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 4gcngaygcnc arccntaygt
20520DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 5atnccrtang cngtrttytt 206543DNAMoraxella
catarrhalis 6atgaaaactt taaaaacact attggcagta tcagcttctt cgttattggc
gatgagtgct 60aacgctgcca tcagctatgg caattctgct gatgctcaac cctatgttgg
tgccaaaatt 120ggtcaagtag acgccaagca aatcaacggt aagaacaccg
cttatggtat ttatgcaggt 180tataactttg accaaaattt tggcgtagaa
cccgaatttg ttggttcaga cgccaaagaa 240tttaatgcag gcgtgagtcc
tgtaaaaggt gatgtgaagt cttttggtgc ttatggcaca 300tatcgctata
acttcatcaa taccccattt tatgccaagg gcaaattagg cattgctaag
360actaaagtag atgttaccag ccgtaatgca actacatact caaacaaaag
cgacaaaacc 420agcctagcag gcggtgttgg tgttggcttt aaaccattag
caaatgtggg cgttgaagca 480agctacaact atctatcaga agatgccaat
gcaattagtt tgggcgctca tttggctttt 540taa 5437180PRTMoraxella
catarrhalis 7Met Lys Thr Leu Lys Thr Leu Leu Ala Val Ser Ala Ser
Ser Leu Leu1 5 10 15Ala Met Ser Ala Asn Ala Ala Ile Ser Tyr Gly Asn
Ser Ala Asp Ala20 25 30Gln Pro Tyr Val Gly Ala Lys Ile Gly Gln Val
Asp Ala Lys Gln Ile35 40 45Asn Gly Lys Asn Thr Ala Tyr Gly Ile Tyr
Ala Gly Tyr Asn Phe Asp50 55 60Gln Asn Phe Gly Val Glu Ala Glu Phe
Val Gly Ser Asp Ala Lys Glu65 70 75 80Phe Asn Ala Gly Val Ser Pro
Val Lys Gly Asp Val Lys Ser Phe Gly85 90 95Ala Tyr Gly Thr Tyr Arg
Tyr Asn Phe Ile Asn Thr Pro Phe Tyr Ala100 105 110Lys Gly Lys Leu
Gly Ile Ala Lys Thr Lys Val Asp Val Thr Ser Arg115 120 125Asn Ala
Thr Thr Tyr Ser Asn Lys Ser Asp Lys Thr Ser Leu Ala Gly130 135
140Gly Val Gly Val Gly Phe Lys Pro Leu Ala Asn Val Gly Val Glu
Ala145 150 155 160Ser Tyr Asn Tyr Leu Ser Glu Asp Ala Asn Ala Ile
Ser Leu Gly Ala165 170 175His Leu Ala Phe180828DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
8ccctatgttg gtgccaaaat tggtcaag 28928DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
9agatgccaag caaatcaacg gtaagaac 281028DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
10gttcttaccg ttgatttgct tggcatct 281128DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
11cttgaccaat tttggcacca acataggg 281218DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
12gcgacaaaac cagcctag 181318DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 13ggtgttggtg ttggcttt
181420DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 14cccctttaaa acatcgccac 201545DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
15ggacgccatg gcaactttaa aaacactatt ggcagtatca gcttc
451645DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 16atcaagctta gtgatggtga tggtgatgaa aagccaaatg
agcgc 451740DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 17gacggcccgg gctggtatca attggcatag
gcggtaagtt 401838DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 18catgctgcag cttgaccaat tttggcacca
acataggg 381940DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 19cactctgcag tagacgccaa gcaaatcaac
ggtaagaaca 402040DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 20gcatgtcgac gtagatgagc tacaaggcgt
gatttgggat 402180DNAArtificial SequenceDescription of Artificial
Sequence Synthetic DNAfragment 21gacgcscarc cstatgttgg tgccaaaatt
ggtcaagtag acgccaagca aatcaacggt 60aagaacaccg cctacggaat 80
* * * * *